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\r\n\tComputer graphics are not entirely an original topic, because it defines and solves problems using some already established techniques such as geometry, algebra, optics, and psychology. The geometry provides a framework for describing 2D and 3D space, while the algebraic methods are used for defining and evaluating equality related to the specific space. The science of optics enables the application of the model for the description of the behavior of light, while psychology provides models for visualization and color perception.
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\r\n\t3D computer graphics (or 3D graphics, three-dimensional computer graphics, three-dimensional graphics) is a term describing the different methods of creating and displaying three-dimensional objects by using computer graphics.
\r\n\tThe first types of graphic interpretations were put in the plane (two-dimensional 2D). Requirements for a universal interpretation led to a three-dimensional (3D) interpretation content. From these creations have arisen applied mathematics and information disciplines of graphic interpretation of content - computer graphics. It relies on the principles of Mathematics, Descriptive Geometry, Computer Science and Applied Electronics.
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\r\n\t3D computer graphics or three-dimensional computer graphics use a three-dimensional representation of geometric data (often in terms of the Cartesian coordinate system) that is stored on a computer for the purpose of doing the calculation and creating 2D images. The images that are made can be stored for later use (probably as animation) or can be displayed in real-time.
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\r\n\tObjects within the 3D computer graphics are often called 3D models. Unlike rendered (generated) images, data that are ""tied"" to the model are inside graphic files. The 3D model is a mathematical representation of a random three-dimensional object. The model can be displayed visually as a two-dimensional image through a process called 3D rendering or can be used in non-graphical computer simulations and calculations. With 3D printing, models can be presented in real physical form.
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\r\n\tComputer graphics have remained one of the most interesting areas of modern technology, and it is the area that progresses the fastest. It has become an integral part of both application software, and computer systems in general. Computer graphics is routinely applied in the design of many products, simulators for training, production of music videos and television commercials, in movies, in data analysis, in scientific studies, in medical procedures, and in many other fields.
Gene therapy involves the insertion, removal or alteration of genes within cells and tissues of a living organism. Although it emerged as a tool to correct hereditary diseases due to a non-functional or missing protein, gene therapy also constitutes an experimental tool to investigate gene function. Since the 1960s, most gene therapy studies are aimed at cancer and hereditary diseases. As a research tool, the most traditional form of gene therapy involves the insertion of a functional gene at a specific location in the host genome in order to replace a mutated or missing gene. This is accomplished by isolating and amplifying the gene of interest, generating a construct containing the necessary elements for the right expression, and inserting this construct into the host organism. Another current approach to the study of gene function involves the genetic engineering of knock-out animals (generally mice), in which a specific gene is turned off through a targeted mutation. The resulting phenotype of a knock-out mouse may provide valuable information regarding the function of the missing gene. However, in some cases the targeted gene is necessary for the embryonic development, in which case the knock-out of that gene is not feasible. Alternatively, individual development may be directed towards compensating for that knock-down gene, in which case the phenotype would not provide much relevant information. Another research tool to study gene function is to create a transgenic organism, which can be a microbe, a plant, a fruit fly, a mouse, a zebrafish, a worm … in which foreign, recombinant DNA (i.e., a transgene) is transferred directly into embryos to result in modified or novel genes. The resulting phenotype can provide valuable information about the role of genes in development, physiology, and disease. Although a transgene integrates in the host cell in a chromosomal location different from the endogenous site, the pattern of expression usually mimics that of the endogenous gene. Transgenic organisms are broadly used for agriculture, production of pharmaceutical drugs and proteins such as insulin, biomedical research, and gene therapy in experimental medicine.
Recent developments have provided us with different kinds of vectors to deliver, remove or modify genes within individual cells and tissues, bypassing undesirable effects of broad knock-down or transgene expression. Basically, vectors used in gene therapy for release of specific genes can be viral or non-viral. Non-viral vectors include naked DNA, oligonucleotides, dendrimers, lipoplexes, and polyplexes, as well as nanoengineered, organically modified silicates. However, whereas artificial DNA transfer methods have very low efficiency and are not useful for some types of cells, natural selection and evolution have led viruses to naturally develop specialized molecular mechanisms to efficiently transport their genomes into the cells that they infect. Delivery of genetic material by a virus is called transduction, and the infected cells are termed as transduced. Actually, the earliest DNA transfer method for bacteria was bacteriophages, which are still commonly used in experimentation. For mammalian cells, the first viral vectors were based on the monkey tumor virus SV40, in which some viral genes can be replaced by foreign genes. Nonetheless, the advantages of viruses like SV40 are limited because only a few genes can be inserted into their genome before the length of the DNA molecule becomes too large to be packed into the viral coat.
Additionally, the genetic material of SV40 is usually degraded after it infects the cells. Therefore, viral vectors experimentally designed for gene therapy are usually created from pathogenic viruses, whose infectivity is much more effective, but they are modified to minimize the biohazard. Modifications usually involve the elimination of part of the viral machinery necessary for their replication, so that the virus can efficiently infect the cells but lacks the necessary proteins for the production of new virions. Experimentally modified viral vectors also have low toxicity, which means minimal unwanted effects on the physiology and viability of the transduced cells. Additionally, lytic viruses, which kill the cells after replicating into them to propagate the infection, cannot be used as vectors. Thus, a limited number of nonlytic viruses modified from the retrovirus, adenovirus (AVV), and adeno-associated virus groups can be used in gene therapy to stably introduce a gene into a cell. Retroviruses insert the genetic material in the form of a RNA molecule, which will produce a DNA copy by the action of the enzyme reverse transcriptase. This viral DNA is efficiently inserted into the host genome as a provirus, where it permanently replicates together with host DNA at each cell division. However, most retroviruses can only infect dividing cells. Lentiviruses (LVVs, like HIV) are a subtype of retroviruses. AVVs and adeno-associated viruses insert their genome in the form of a DNA molecule. Adeno-associated viruses, so named because they need the help of an adenovirus for replication, are small viruses whose genome consists of a single stranded DNA molecule. Once the adeno-associated virus enters the nucleus, their single-stranded DNA becomes double-stranded and integrates into the host genome, like retroviruses. However, AVVs enter the nucleus but do not replicate, remaining as extrachromosomal double-stranded DNA molecules. As many differentiated cells in the brain and other tissues lack the capability to divide, LVVs, AVVs, and adeno-associated viruses are acquiring increasing popularity in gene therapy applications because they can transduce non-dividing cells.
Currently, gene therapy is mainly being focused on the study of genetic disorders such as cancer, diabetes mellitus, cardiovascular diseases and nervous system pathologies, among others. More specifically, regarding the applications in the nervous system, this chapter will highlight the potential of viral vectors as tools for the investigation of the role of altered proteins in neuropathological processes and review our recently published findings (Sunico et al., 2008; 2010; Sunico & Moreno-López, 2010; Montero et al., 2010) in the context of the existing literature. Briefly, we have used AVVs and LVVs to study the function of 2 dysregulated proteins in pathological events occurring at the peripheral (nerve) and central (motoneuron) levels after the severe crushing of a motor nerve in adult rats. The use of AVVs is proposed as a feasible gene therapy strategy for the enhancement of peripheral nerve regeneration in acquired peripheral neuropathies. Additionally, our outcomes support the promising benefits of gene therapy for the delivery of diverse genetic elements into specific populations of brain cells to treat several neurodegenerative disorders.
Most neurodegenerative disorders and prion diseases have common cellular and molecular mechanisms, including dysregulation of protein expression, function and/or aggregation. Alteration in the expression level of nitric oxide (NO) synthase (NOS) is a hallmark of Alzheimer (AD), Parkinson (PD), and Huntington diseases (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and HIV dementia in humans and/or in animal models, as well as after peripheral and central traumatic lesions of the nervous system (Montero et al., 2010; Moreno-López et al., 2011). Subsequently, dysregulation of protein expression and/or function is a key event in a broad spectrum of neuropathological states. Unmasking the role of imbalanced proteins in anatomo-functional alterations of neurons in neurodegenerative disorders deserves attention. Our research has shown that viral vectors may be useful tools to explore the role of altered proteins in neuropathological processes. Using AVVs and LVVs, we studied the role of 2 NOS isoforms in pathological events occurring at the peripheral nerve and at the motoneuron after XIIth nerve crushing in adult rats.
NO is a short-lived, bioactive free radical which, as a gaseous molecule, freely crosses plasma membrane. NO and L-citrulline are the products of hydroxylation of a guanidine nitrogen of L-arginine and subsequent oxidization of the Nω-hydroxy-L-arginine intermediate by NOS, a heme-containing enzyme that utilizes tetrahydrobiopterin (H4B) as a redox cofactor. Electron transfer reactions carried out by NOS are regulated by a Ca2+-binding protein (calmodulin). NOS also needs NADPH as an electron donor and requires molecular oxygen to carry out the reaction. In their active form, the known NOS enzymes form dimers in which each NOS monomer is associated with a calmodulin molecule. Three major isoforms of NOS have been identified, coded by different genes and differing in localization, regulation, catalytic properties, and inhibitor sensitivity. The nNOS isoform was the first to be purified and cloned (also known as NOS-I) and is predominantly found in the neuronal tissue. Through their specialized postsynaptic density-95/disks large/zona occludens-1 domains, nNOS can physically associate with postsynaptic density protein-95 (PSD-95). In turn, PSD-95 binds to motifs in the C-terminus of NMDA receptor NR2 subunits. These molecular interactions may provide the mechanistic basis for a functional coupling between Ca2+ influx through NMDA receptors and NO production. The iNOS isoform (also known as NOS-II) is inducible in a wide range of cells and tissues, including activated macrophages and, astroglia and microglia in the pathological CNS. This isotype is Ca2+-independent and always catalytically active when expressed. However, iNOS is not the only isoform induced in pathological conditions. Finally, eNOS (or NOS-III) is the primary isoform found in vascular endothelial cells (Moreno-López et al., 2011).
Injured nerve fibers in the peripheral nervous system maintain the capacity to regenerate even over long distances in adult mammals. However, after nerve transection, stumps of damaged nerves must be surgically joined to guide regenerating axons into the distal nerve stump. Even so, severe functional limitations persist after restorative surgery. Since most previous studies have used nerve transection, it is important to signal distinctions between different forms of nerve lesion. Regenerative and survival capacities could be differentially compromised in adult motoneurons following transection or avulsion of the peripheral nerve. However, crushing of a motor nerve causes an immediate and complete suppression of neuromuscular connectivity but, unlike transection, it preserves the endoneural tube, thus providing neurotrophic support and a physical guide for the growing proximal axonal endings (Moreno-López, 2010). A few weeks after nerve crushing, muscle re-innervation takes place without significant neuronal loss. Therefore, traumatic injury by crushing is a plausible model for the identification of molecules that regulate degenerative and regenerative processes after acquired peripheral neuropathies.
We have developed a model of acquired peripheral motor neuropathy induced by a traumatic insult that causes a well-characterized range of functional and synaptic impairments in the insulted motoneurons. This experimental model has been useful to test the role and effectiveness of diverse factors in degenerative/regenerative processes after nerve injury by means of functional and anatomical approaches. The hypoglossal system -hypoglossal motoneurons (HMNs), XIIth nerve and their target muscle (the tongue)- has been well characterized by our group in physiological and pathological conditions (González-Forero et al., 2004; Montero et al., 2008; 2010; Sunico et al., 2005; 2008; 2010; 2011). This motor system offers several advantages, since muscle and motoneuron activities can be accurately measured and its peripheral and central partners are easily accessible to carry out damage strategies, local microinjections and electrophysiological recordings.
Crushing the XIIth nerve immediately induces a complete suppression of neuromuscular connectivity, demonstrated by the complete absence in the tongue of the compound muscle action potential (CMAP) evoked by electrical stimulation of the injured nerve at the proximal portion (Fig. 1). CMAP was completely absent for 1, 3 or 7 days after crushing, even when supramaximal nerve stimulation was applied. These results indicate that our method of nerve crushing effectively disconnected most HMNs from their targets for at least 1 week. Evidence of muscle re-innervation was obtained at 15 days after crushing. Subsequently, there was a slow and progressive recovery of CMAP and at 30 days after crushing its amplitude was similar to the control condition (Fig. 1).
In the hypoglossal nucleus (HN), most motoneurons discharge bursts of action potentials synchronized with the inspiratory phase of breathing (Fig. 2). This characteristic activity persists even after animal decerebration and/or under anesthesia. The main alterations observed in the firing pattern of HMNs following XIIth nerve crushing were an overall reduction in firing rates and an almost complete loss of modulation by chemosensory afferents. The inspiratory activity of motoneurons was modulated by chemoreceptor-driven changes in response to alterations in end tidal CO2 (ETCO2). The activity bursts of HMNs increased when ETCO2 rose and decreased when ETCO2 declined. One week after XIIth nerve crushing the chemosensory-mediated responsiveness of HMNs to ETCO2 changes decreased (Fig. 2). Synaptic stripping of HMNs after nerve injury is at least partially responsible for reduced response of motoneurons to chemoreceptor-modulated inspiratory drive (Fig. 2).
Experimental design and time course of neuromuscular function recovery after XIIth nerve crushing. (a) To evaluate the time course of the neuromuscular function recovery, the compound muscle action potential (CMAP), evoked by electrical stimulation (St.) of the XIIth nerve, was recorded using electrodes implanted in the genioglossus muscle. Inset: Photomicrograph of a sagittal section at the level of the crushing site extracted 3 h after injury, stained by neutral red. Note that axons are fully transected but endoneural tube is preserved. Scale bar: 500 μm. (b) CMAPs evoked in the genioglossus muscle by single shock stimulation (arrowheads signal stimulus artifacts) of XIIth nerve in control and at 7, 15, 22, 30 and 45 days post-lesion. For comparison, CMAPs evoked by left (L, intact side) and right (R, crushed side) XIIth nerve stimulation are illustrated. Each trace represents an average of 10 individual traces. Dotted lines at 15 and 22 days represent how the Δ latency was calculated. Gray traces at 15 days represent the responses obtained in the same animal after injection of a neuromuscular blocker gallamine triethiodide in the genioglossus muscle. Note complete absence of CMAP on day 7 and the first signs of muscle re-innervation 15 days post-injury. Figure modified from Sunico et al., 2008 and Sunico & Moreno-López, 2010 (inset in (a)). © 2008 Elsevier Ltd and © 2010 Elsevier Ireland Ltd, respectively.
XIIth nerve crushing induces a reduction of HMNs CO2-modulated response and of their synaptic coverage. (a) Schematic diagram of the experimental preparation. Unitary discharge activity of HMNs and expired CO2 were obtained in decerebrated, vagotomized rats, which had been injected with a neuromuscular blocking agent and lightly anesthetized. Right, characterization of firing properties of HMNs. From top to bottom, traces represent the extracellularly recorded spike discharge for a control inspiratory HMN, the instantaneous firing rate (FR, in spikes/s) and the partial pressure of CO2 as a percentage of the expired air. Mean firing rate (mFR) per burst was measured and analyzed in relation to simultaneous ETCO2 measurements (dotted line in the middle trace). End-tidal CO2 (ETCO2) is indicated on the CO2 record as a dashed line. (b) Illustrative examples of the time courses of the mean firing rate modulation (mFR, in spikes (sp)/s/burst; right y-axis; gray trace) relative to ETCO2 levels (left y-axis; black trace) for a control HMN and a motoneuron recorded 7 days after XIIth nerve crushing. (c) Confocal high-magnification photomicrographs of HMNs identified by the presence of a tongue-injected retrograde tracer (FluoroGold; green) in sections immunostained for the synaptic marker synaptophysin (red) obtained from control animals or 7 days after XIIth nerve crushing. Scale bars: 10 μm. Figure modified, with permission, from Gonzalez-Forero et al., 2004 (a); Sunico et al., 2005 (c) and Montero et al., 2010 (b). © 2004 The Physiological Society, © 2005 Society for Neuroscience and © 2010 The Physiological Society, respectively.
Up-regulation of NOS also happens as a consequence of different kinds of peripheral neuropathies. More specifically, after traumatic injury of a motor nerve, NOS expression is up-regulated at 3 different levels: in the damaged nerve, in the denervated muscle, and centrally at the injured motoneurons. In the distal stump of the affected nerve, all 3 major isoforms of NOS are up-regulated: iNOS is de novo expressed in dedifferentiated Schwann cells and in infiltrated macrophages, whereas nNOS is accumulated in the growing motor axons. However, eNOS, which is constitutively found in blood vessels, is overexpressed in vasa nervorum of the distal stump and around the injury site after the damage (Moreno-López, 2010). At the central level, nNOS, but not iNOS, is expressed de novo in the soma of motoneurons after traumatic motor nerve injury (Sunico et al., 2005). nNOS is then outwardly transported, accumulating in growing axons at the peripheral level (Moreno-López, 2010). However, eNOS induction has not been reported in injured motoneurons so far (Montero et al., 2010).
Evidences of NO as a harmful molecule for nerve regeneration show that systemic administration of Nω-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, improved motor nerve regeneration in mice after sciatic nerve transection (Zochodne & Levy, 2005). L-NAME also protected motoneurons from death and substantially increased the number that re-innervated the target muscle after facial nerve transection and repair, accompanied by improved motor function recovery (Wang et al., 2009). In the same direction, our studies showed that L-NAME treatment advanced the onset of neuromuscular reconnection after hypoglossal nerve crushing (Sunico et al., 2008). However, the pattern and time course of expression of each NOS isoform (Moreno-López, 2010) suggest that the NO from different origins could regulate diverse degenerative/regenerative processes after peripheral injury in a time-dependent manner. In nNOS or iNOS knockout mice, peripheral recovery was impaired after nerve injury due to delayed Wallerian degeneration, axon breakdown and Schwann cell reaction in the distal stump (Zochodne & Levy, 2005). Nevertheless, axon counts, myelination, and recovery of sensory and motor function in eNOS knockouts and wild-type mice were comparable after transection and reconstruction, although a delay of 2 days in revascularization was observed in eNOS knockout mice (Zochodne & Levy, 2005). The inherent risk of compensatory mechanisms for the disrupted enzyme during brain development is widespread in knockout animals. This may occur though a normally present redundant pathway and may be due to differences in expression of alternatively spliced isoforms of a particular NOS. For example, NOS catalytic activity levels in the brain of nNOS knockout mice are persistently low, which may mask the effect of nNOS deficiency (Moreno-López, 2010). Therefore, results obtained from knockouts, although useful, must be interpreted with caution.
Systemic application of relatively isoform-specific NOS inhibitors is another strategy that has been used to study the role of each isoform in degenerative/regenerative processes after nerve injury. nNOS or iNOS inhibition by the relatively specific inhibitors for nNOS 7-nitroindazole (7-NI), S-methyl-L-thiocitrulline (SMTC) and iNOS L-N6-(1-iminoethyl)-lysine hydrochloride (L-NIL), has contributed contradictory results. Whereas systemic administration of 7-NI, SMTC or L-NIL had beneficial effects on axonal regeneration after facial nerve transection and repair (Wang et al., 2009), positive effects were not obtained after XIIth nerve crushing by treatment with 7-NI or aminoguanidine (AG), another iNOS inhibitor (Sunico et al., 2008). Strikingly, administration of the relatively specific eNOS inhibitor L-N(5)-(l-iminoethyl)ornithine (L-NIO) accelerated the onset of muscle re-innervation after hypoglossal nerve crushing by promoting axonal regrowth (Sunico et al., 2008). Contradictions can be explained by differences in the lesion model and in the dose and relative specificity of NOS inhibitors (Moreno-López, 2010). In any case, these results pointed to eNOS as the major source of detrimental NO in axonal regeneration, at least in the first week after nerve injury.
In general, a strong consensus exists stating that NOS inhibition, at least during the first week after traumatic motor neuropathy, accelerates and improves motor function recovery. However, the administration route must be carefully taken into account. NO has important roles in the immunological, cardiovascular and nervous systems. Key physiological functions, such as learning, platelet aggregation, arterial blood pressure, and immune responses, could be disrupted by chronic and systemic NOS inhibition. NOS inhibition as a plausible treatment to improve functional recovery after nerve injury must bypass unwanted side effects of systemic administration of the inhibitors. The therapeutic strategy should entail specific inhibition of the proper NOS isoform that is detrimental to nerve regeneration and the selected NOS inhibitor should be locally administered (Moreno-López, 2010). Endothelial isoform has been described as having negative effects on functional recovery in the traumatic motor nerve injury model (Sunico et al., 2008), mainly inhibiting axonal regrowth. Due to the apparent involvement of eNOS in vasodilation of blood vessels and platelet aggregation, however, chronic systemic treatment with a specific eNOS inhibitor is not advised. Such a treatment can affect cardiovascular function, inducing hypertension and increasing the risk of thrombosis and atherosclerosis. Finally, systemic administration of NOS inhibitors is not advised because they cause motor deficits in mice and rats (Moreno-López, 2010). Feasible tools to improve motor function recovery after nerve injury without systemic side effects could involve local application of viral vectors. Promising results have been obtained by our group, which has reported that intraneural administration of an AVV expressing the dominant negative of eNOS (AVV-TeNOS) accelerated and improved motor function recovery after XIIth nerve crushing (Sunico et al., 2008).
To elucidate the role of eNOS, recombinant protein expression has been induced by viral transfection. There are different approaches to induce a full or partial “loss of function” using viral transgenesis. In this chapter we illustrate the use of a dominant negative protein, which interferes with the target protein or its function. To suppress eNOS activity, a recombinant AVV was used to express a truncated form of eNOS (TeNOS) under the control of the human cytomegalovirus (hCMV) promoter (Kantor et al., 1996). TeNOS, which lacks catalytic activity, acts as a dominant negative inhibitor of wild-type eNOS by heterodimerization with the native protein (Liu et al., 2011). As a viral control, a recombinant AVV was used to express the enhanced green fluorescent protein (eGFP) directed by the hCMV (AVV-eGFP). This gene therapy strategy successfully revealed the involvement of eNOS from the nucleus tractus solitarii in hypertension of spontaneously hypertensive rats (Kasparov et al., 2004; Waki et al., 2003; Waki et al., 2006; Paton et al., 2007). The expression of proteins under control of the hCMV promoter is characterized by a very rapid increase in expression, peaking 10 hr after transfection (Stokes et al., 2003). Furthermore, when AVV-eGFP and AVV-TeNOS were injected in the nervous system, eGFP and TeNOS were expressed in glia and endothelial cells 7 days after transduction; expression diminished greatly at 14 days after vector injection (Waki et al., 2006). Therefore, a single intranerve injection of AVV-TeNOS could be expected to provide effective chronic inhibition of eNOS for at least 1 week in the transduced cells. It is noteworthy that this AVV did not retrogradely transduce motoneurons when injected into the nerve (Sunico et al., 2008; Moreno-López, 2010).
A single intranerve microinjection (3 μl) of AVV-TeNOS (2.2 x 1010 infective units/ml) on the day of the XIIth nerve crushing accelerated the recovery of neuromuscular transmission, resulting in a measurable CMAP 1 week after the damage (Fig. 3). Furthermore, the CMAP was fully recovered 22 days after the damage, which is 1 week earlier than in the untreated or eGFP transduced animals (Sunico et al., 2008). Stability of recovery was confirmed by the recording of a control-like CMAP from animals administered with AVV-TeNOS and studied at 62 days post-injury (Fig. 3). These results demonstrated that a single intraneural injection of the AVV-TeNOS efficiently accelerates the functional recovery of the neuromuscular junction after nerve injury (Sunico et al., 2008).
In addition, axonal regeneration was increased by AVV-TeNOS administration, compared with the AVV-eGFP treated group. Specifically, the number of retrogradely labeled motoneurons at 2 days after nerve crushing and intraneural injection of AVV-eGFP was significantly lower than that observed with AVV-TeNOS (Fig. 3). The number of retrogradely labeled HMNs at 7 days post-lesion in AVV-TeNOS-injected rats was similar to the control condition illustrating that AVV administration did not affect motoneuron viability (Sunico et al., 2008). Therefore, local eNOS inhibition with AVV-TeNOS speeds up neuromuscular functional recovery that is associated with accelerated axonal regeneration, among other possible mechanisms.
Schwann cells (SC) play a very important role in promoting axonal regeneration through the distal stump. After peripheral nerve axotomy, Wallerian degeneration involves axonal degradation as well as myelin breakdown and clearance in the distal stump of the injured nerve. For the subsequent axonal regeneration, SC dedifferentiation is required (Jessen & Mirsky, 2008). Thus, following nerve axotomy, SCs change from a myelin-producing state to a dedifferentiated, proliferating non-myelin-forming state (Stoll & Muller, 1999). In this dedifferentiated state, SCs are characterized by the formation of strands called bands of Bungner, which are the cellular substrate to guide growing axons, together with the up-regulation of several regeneration associated proteins, such as cell adhesion molecules, cytokines, neurotrophins, and growth factors that promote axonal regeneration (Araki et al., 2001; Stoll & Muller, 1999). More specifically, the growth-associated protein 43 kDa (GAP-43) is strongly up-regulated in regenerating axons and in dedifferentiated SCs (Curtis et al., 1992; Plantinga et al., 1993; Scherer et al., 1994). As soon as 1 day after transection or crushing of sciatic nerve, GAP-43 mRNA is significantly increased in the distal stump, remaining at high levels for at least 4 weeks (Plantinga et al., 1993). Thus, GAP-43 protein, which is involved in cell shape plasticity and motility (Curtis et al., 1992; Scherer et al., 1994), contributes to SC reorganization to form bands of Bungner in the degenerative/regenerative processes that take place after nerve injury.
Chronic intraneural inhibition of a dominant negative for eNOS using an AVV accelerates neuromuscular function recovery and axonal regeneration. (a)-(c) CMAPs evoked in the genioglossus muscle by single shock stimulation (arrowheads point to the stimulus artifact) of XIIth nerve at 7 (a), 22 (b), or 62 (c) days after intraneural injection of AVV-eGFP or AVV-TeNOS. For comparison, recordings obtained by stimulation of the left (intact) and right (crushed) XIIth nerve are illustrated. Each trace represents an average of 10 individual responses. (d), (e) Photomicrographs of coronal sections of the right HN showing FluoroGold-labeled motoneurons in animals injected with AVV-eGFP (d) or AVV-TeNOS (e) on the crushing day. FluoroGold was applied on day 2 post-crushing and the animals were perfused 7 days after retrograde marker application. Scale bar = 100 µm. Figure modified from Sunico et al., 2008. © 2008 Elsevier Ltd.
Given that AVV-TeNOS intraneural injection speeds up motor function recovery after XIIth nerve injury (Sunico et al., 2008), SCs were targeted to find out a feasible mechanism by which eNOS inhibition could enhance nerve regeneration, with the presumption that endothelial NO could be acting as a negative regulator of SC dedifferentiation after nerve crushing. To approach this issue, GAP-43 protein was analyzed in this study as a marker of regenerative processes, together with the density of SCs and bands of Bungner, in injured nerves after intraneural inhibition of eNOS. Indeed, our results indicate that endothelial NO confers a delaying action on SC dedifferentiation. As soon as 2 days following crushing of XIIth nerve together with AVV-TeNOS microinjection in the distal stump, GAP-43 immunoreactivity increases compared to control treatment with AVV-eGFP (Fig. 4). The reason for this, at least in part, could be an increase in GAP-43-positive cells, presumably SCs, after eNOS inhibition. Formation of bands of Bungner is also favored by TeNOS (Sunico & Moreno-López, 2010). Thereafter, all these data indicate that eNOS inhibition enhances the number of SCs and the subsequent formation of bands of Bungner after peripheral nerve injury (Fig. 4).
To summarize, negative regulators for myelination, which activates the SC dedifferentiation program, involve many extrinsic and intrinsic signals (Jessen & Mirsky, 2008). It is a well established fact that injury-related signals from neighboring SCs and neurons accelerate and influence SC dedifferentiation in vivo (Jessen & Mirsky, 2008). However, these recent studies also identify endothelial NO as a negative regulator of SC dedifferentiation, thus pointing to the vascular system as a source of signals that regulate some of the degenerative/regenerative processes that take place after peripheral nerve injuries (Sunico & Moreno-López, 2010). For this reason, the local endothelium is cautiously proposed in this chapter as a gene therapy target in the acute events that take place in peripheral neuropathies, and virally mediated eNOS antagonism is highlighted as a viable novel therapeutic strategy.
TeNOS transgene increases the number of GAP-43 cells and bands of Bungner after XIIth nerve crushing. (a)-(c) GAP-43-immunoreactive (a) cells co-identified by Hoechst nuclear staining (b); the average number of GAP-43-positive cells (in thousands per cubic millimeter) at 4 and 5 mm distal to the injury site receiving the indicated adenoviruses is plotted (c). (d) Confocal high-magnification photomicrograph showing GAP-43-positive SCs forming two bands of Bunger disposed in parallel. (e)-(g) Confocal micrographs illustrating that after AVV-TeNOS injection the re-organization of SCs in bands of Bungner was more evident than in AVV-eGFP-treated injured nerves. The average number of bands of Bungner (in thousands per cubic millimeter) at 4 and 5 mm distal to the injury site receiving the indicated adenoviruses is plotted (g). Scale bars: (a), (b), (d), 10 μm; (e), (f), 50 μm. *P < 0.05; non-parametric Mann-Whitney U test. Figure modified from Sunico & Moreno-López, 2010. 2010 Elsevier Ireland Ltd.
Disconnection of motoneurons from their target myocytes interrupts mutual trophic relations, leading to deep alterations in the structural and physiological properties of both motoneurons and muscle fibers. Axotomy provokes changes in axonal, synaptic and intrinsic membrane properties, including enhanced somato-dendritic excitability, decreased axonal conduction speed, considerable loss of afferent synaptic contacts, and disorders in the firing properties and recruitment order of motor units (González-Forero et al., 2004; 2007). Synapse loss is the major feature underlying cognitive impairment in patients and/or animal models of AD, PD, MS, HD, and HIV-related dementia (Sunico et al., 2010; Moreno-López et al., 2011). Interestingly, synaptic alteration, rather than neuronal death, is the main factor responsible for the age-related downturn in neuronal function. Synapse loss also happens in several motor maladies, including ALS, progressive muscular atrophy, and traumatically-injured motor axons (Sunico et al., 2005; 2010). Interestingly, de novo expression of nNOS in motoneurons commonly occurs in response to the physical injury of a motor nerve and in the course of ALS. In both conditions, this event precedes synaptic withdrawal from motoneurons (Moreno-López et al., 2011). Changes in functional properties of injured cells also were prevented using various pharmacological agents targeting nNOS. NO-mediated disturbances involve changes in intrinsic membrane properties and anatomical synaptic deterioration that suggest a major pathological role of nNOS. However, nNOS is only one of the numerous proteins dysregulated after nerve damage. Therefore, the actual role for nNOS is less clear within the complex scenario created by multiple dysregulated proteins (Montero et al., 2010).
In an attempt to scrutinize whether nNOS up-regulation is sufficient to promote alterations after axonal injury on motoneurons, we have virally induced de novo expression of nNOS in non-axotomized HMNs together with complementary down-regulation of nNOS expression using virally mediated gene knock-down (Montero et al., 2010; Sunico et al., 2010). Replication-deficient recombinant AVVs (1010-1011 infective units/ml), directing the expression of enhanced green or monomeric red fluorescent proteins (eGFP and mRFP, respectively) or nNOS, were injected into the tongue to retrogradely transduce HMNs (Fig. 5). AVVs expressed eGFP, mRFP, or nNOS downstream the hCMV promoter. The AVV-eGFP and AVV-mRFP vectors were used as controls to test virally induced side effects.
Neonatal HMNs were retrogradely transduced by injecting AVV-nNOS and/or AVV-eGFP into the genioglossus muscle. Ratiometric real-time NO imaging was used to record NO released around transduced HMNs in response to a glutamatergic stimulus (Fig. 5). For that purpose, the NO-sensitive fluorescent probe 1,2-diaminoanthraquinone sulphate (DAA) was perfused together with the reference dye Alexa 633. We found that the slope of DAA fluorescence increased around eGFP/nNOS-transduced motoneurons in response to glutamate, which was prevented by adding the NOS inhibitor L-NAME to the bath. This increase of NO created a gradient of concentration around the transduced HMNs, which in brain parenchyma showed a space constant of 12.3 µm. Thus, HMNs retrogradely transduced with AVV-nNOS express a functional enzyme that synthesizes NO in response to glutamatergic stimulation (Montero et al., 2010).
Retrograde cotransduction of adult HMNs, after injection in the tongue of AVV-eGFP/AVV-nNOS, induced a significant reduction in their synaptic coverage, as shown by immunohistochemistry and electron microscopy. Thus, nNOS transfection mimicked the effect of axonal injury on the synaptic coverage of HMNs. In this way, it seems clear that NO, synthesized by up-regulated nNOS in adult axotomized motoneurons, is not only necessary but also sufficient to trigger the molecular cascade leading to synapse withdrawal from HMN perikarya (Sunico et al., 2010; Moreno-López et al., 2011).
AVV transfection of HMNs with functional nNOS. (a) Left, AVV injection into the tip of the tongue retrogradely transfected HMNs. Right, an illustrative example of a retrogradely cotransduced HMN after injection in the tip of the tongue of AVV-eGFP+AVV-mRFP. (b) Merged DIC and eGFP channels showing the tip of a pipette (arrow) ejecting dyes close to an eGFP-transfected HMN. The circle indicates the region of interest (ROI) used to construct the plot illustrated in (c). (c), (d) Time courses of the DAA/Alexa 633 ratio within a 5 μm diameter ROI placed just at the border of transfected HMNs by means of the indicated AVVs before and after addition to the perfusate of L-glutamate (500 μM; arrows). Slopes of the regression lines adjusted before (Sbglut) and after (Saglut) glutamate addition are indicated in (d). The slope increase after Glut application in (d) result from a rise in DAA relative to Alexa fluorescence. This indicates that NO synthesized by transduced HMN interacts with the NO-sensitive dye DAA. (e) Averaged Saglut–Sbglut was obtained from HMNs transfected with the specified AVVs and incubated with the indicated drugs. Drugs were added to the bath 5 min before glutamate. Inset, illustration signaling the location of ROIs analyzed per motoneuron. The average of Saglut–Sbglut was taken as the representative HMN value. Prevention of the slope increase after Glut by pre-incubation with the NOS inhibitor L-NAME strongly suggests that the change in the slope is mediated by generation of NO. (f) NO gradient in brain tissue surrounding nNOS expressing HMNs. Average Saglut–Sbglut was obtained from HMNs transfected with the indicated AVVs relative to the distance from the center of the ROI to the motoneuron border. Measures have been normalized relative to the value obtained in the ROI nearest to the motoneuron. Inset illustrates how ROIs were located for this type of measure. (g) Data presented in (f) were well fitted to the exponential decay equation used to calculate the theoretical space constant of the NO gradient created around AVV-nNOS transfected motoneurons. Scale bars: 50 μm. Figure modified, with permission, from Montero et al., 2010 (a) and Sunico et al., 2010 (b-g). © 2010 The Physiological Society and Society for Neuroscience, respectively.
For functional studies we injected AVV directly into the HN (Fig. 6). Five to 7 days after AVV administration, numerous neurons were identified as positively transfected at the injection site. Besides, a high number of astrocytes were also transfected, as confirmed by co-immunostaining against the astroglial cell marker glial fibrillary acidic protein (GFAP). Inspection of brainstem slices did not reveal eGFP-positive cell bodies in areas that project to the HN, such as the ventrolateral reticular formation (VLRF; Fig. 6). This argues against the possibility that our results could be affected by retrograde spread of AVV from the injection
Specificity of viral injections and transfections. (a) Schematic diagram of viral administration. AVV and LVV were administered into the HN. (b) Epifluorescence photomicrograph of a coronal section at the level of the HN obtained 6 days after intranuclear injection of AVV-eGFP. Note that injection was almost fully restricted to the HN; cc, central canal. (c) Photomicrograph of a coronal section obtained 7 days after viral administration at the indicated distance from the injection site showing a broad number of eGFP-expressing hypoglossal neurons. At this level a high frequency of co-transfection was observed after intranuclear injection of AVV-eGFP/AVV-mRFP (right panels). (d) Low magnification photomicrograph of a coronal section at the level of AVV-eGFP injection site, obtained 7 days after viral administration. Note the absence of infected neurons in neighboring regions, such as the ventrolateral reticular formation (VLRF), which project directly to the HN. Asterisk points to the mark that identifies the right side of the brainstem. (e), (f) Photomicrographs from coronal sections at the level of the injection place obtained 7 days after intranuclear injection of LVV-miR-shRNA/nNOS in untreated animals (e) and those receiving doxycycline (Dox) in the drinking water (f). Scale bars: (b), (e), (f) 250 μm; (c), (d) 500 μm; (c) right panels 100 μm. Figure modified from Montero et al., 2010. © 2010 The Physiological Society.
site. A large number of eGFP-positive neurons were identified, strictly within the HN, 200-300 μm rostrocaudal and ipsilateral to the injection site (Fig 6). Furthermore, astroglia was not transduced at these remote locations, as colocalization of eGFP and GFAP was absent there (Montero et al., 2010). Therefore, functional tests were performed in these areas away from the injection site to minimize any contaminating effects of nNOS transduction of astrocytes.
Unilateral AVV-nNOS microinjection in the HN of adult rats induced axotomy-like changes in HMNs such as alterations in axonal conduction properties and reduction in the responsiveness to synaptic drive (Montero et al., 2010). In AVV-eGFP-transfected animals, as in controls, the majority of HMNs display a characteristic respiratory pattern of bursts of action potentials that is synchronized with the inspiratory stage of breathing. In contrast, the mean firing rate in basal conditions (ETCO2=4.8-5.2%) was considerably reduced after intranuclear microinjection of AVV-eGFP/AVV-nNOS (Fig. 7), likewise 1 week after hypoglossal nerve damage (Gonzalez-Forero et al., 2004). This reduction in the mean firing rate was prevented by chronic treatment with L-NAME or 7-NI, a relatively specific nNOS inhibitor. These results suggest that the reduction in the mean firing rate is evoked by NO synthesized by transduced nNOS in the HN.
To gain specificity in nNOS activity inhibition, we used a neuron-specific LVV to knock-down nNOS. This LVV system has been recently described (Liu et al., 2008; 2011). Briefly, for nNOS knock-down, we used a binary LVV system that requires co-operative action of 2 viral vectors. The first vector expresses tetracycline-sensitive transactivator Tet-off under control of an enhanced synapsin-1 promoter (Liu et al., 2008; 2011). The second LVV harbors an expression cassette for a miRNA30 (miR30)-based short hairpin (shRNA) interference system (Stegmeier et al., 2005) under control of a Tet-sensitive promoter. The system expresses a miRNA30-like hairpin targeting the gene of choice as a fusion with the eGFP, which facilitates targeting of the RNA duplex into the RNA-induced silencing pathway. Tet-off is able to bind tetracycline or similar molecules such as doxycycline (Dox); this renders it unable to bind to the Tet-sensitive promoter and blocks the expression of the hairpin (Fig. 6). This system is then referred to as LVV-miR-shRNA/nNOS.
nNOS-induced effects on mean firing rate were fully prevented by administration of LVV-miR-shRNA/nNOS before AVV-nNOS injection into the HN, indicating that nNOS expression in neurons, but not in glial or endothelial cells, affects mean firing rate (Fig 7).
In physiological conditions, the activity of HMNs increases when the ETCO2 rises, and decreases when the ETCO2 lowers. Axonal injury of HMNs induces a decrease in their response to chemoreceptor-modulated inspiratory drive (see Fig. 2; González-Forero et al., 2004) which was NO-mediated (Sunico et al., 2005). Strikingly, when HMNs are transduced with transgenic nNOS, their sensitivity to the chemoreceptor-modulated inspiratory drive is dramatically decreased, an alteration that was equivalent to the effects of crushing on motoneuron sensitivity to their afferent drive (Fig. 8). Chronic administration of L-NAME or 7-NI, as well as LVV-miR-shRNA/nNOS injection, protected against the changes in motoneuron sensitivity induced by de novo expression of nNOS. Altogether, these findings suggest that nNOS transgene-derived NO is sufficient to activate the molecular processes that lead to a decrease in motoneuron sensitivity to their afferent inputs.
NO-mediated synaptic withdrawal from motoneurons is an underlying factor that is at least partially responsible for functional changes induced by axonal injury of motoneurons (Gonzalez-Forero et al., 2004; Sunico et al., 2005; 2010). We have recently reported that de novo synthesis of NO, induced by retrograde transduction of HMNs through muscle injection of AVV-nNOS, was sufficient to induce a withdrawal of synaptic boutons on HMNs (Sunico et al., 2010). This effect came together with a strong decline in the evoked excitatory postsynaptic potential on motoneurons in vitro (Sunico et al., 2010). Additionally, AVV-nNOS microinjection into the HN induced a reduction in the number of synaptic boutons apposed to HMNs, which could be prevented by preceding injection of LVV-miR-shRNA/nNOS (Fig. 8). Taken together, these observations indicate that main functional alterations induced in motoneurons by nNOS transgene expression or axotomy involve NO-directed synaptic re-arrangements.
AVV-nNOS injection into the HN mimics effects of XIIth nerve crushing on the basal firing activity of the HMNs. Representative examples show the discharge activity of HMNs recorded at basal conditions (ETCO2 = 4.8–5.2%) at the indicated conditions. For each panel, traces are the raw signals (top) of extracellularly recorded spike activity and the histogram of instantaneous firing rate (in spikes (sp)/s; bottom). Note that AVV-nNOS induced alterations in basal activity of HMNs were prevented by 7-NI or LVV-miR-shRNA/nNOS. Figure modified from Montero et al., 2010. © 2010 The Physiological Society.
In summary, we suggest that de novo expression of nNOS creates a repulsive gradient of NO around the motoneurons in an activity-dependent manner that can alter their inherent membrane properties as well as their synaptic coating. These results further point to nNOS as a pivotal target for the development of tools for the treatment of peripheral neuropathies and neurodegenerative disorders characteristically accompanied by central up-regulation of nNOS. Additionally, this opens a line of research for a strategy to elucidate the role of dysregulated proteins in the neuronal impairment taking place in the course of several neuropathological situations.
AVV-nNOS injection into the HN mimics effects of XIIth nerve crushing on the chemoreceptor-modulated inspiratory activity of HMNs and on their synaptic coverage. (a) Illustrative examples of the time courses of the mean firing rate modulation (mFR, in spikes (sp)/s/burst; right y-axis) relative to ETCO2 levels (left y-axis) for HMNs recorded 7 days after the injection of the indicated viruses into the HN. Figure modified from Montero et al., 2010. © 2010 The Physiological Society.
Dysregulation of protein expression in specific cell populations of the central nervous system is a common hallmark in most neurodegenerative diseases. Ideally, this problem could be approached by somatic gene delivery targeted to specific cell types within a certain nucleus in order to increase or decrease the expression of a particular gene, with a precise control over the temporal expression of the transgenes. Transfection of cell lines with plasmids is a very efficient process, mainly thanks to the use of specific chemical reagents that increase the permeability of the cells. However, transfer of genes into a brain in vivo is still a challenging task. Nonetheless, gene delivery into brain cells using viral vectors has been successfully performed, even for long-term gene expression (Thomas et al., 2003; Papale et al., 2009). Likewise, viral vectors have been successfully used both in vitro and in vivo for the delivery of new genomic tools such as small interference RNAs (Snove and Rossi, 2006; Paddison, 2008).
As supported by our outcomes using an experimental model of peripheral neuropathy, the re-establishment of a functional gene through the insertion, removal, or modification of genes within cells and tissues is a very promising therapeutic alternative to chemicals for the treatment of human genetic disorders. Unfortunately, although in theory it could be considered as the holy grail, in practice each type of gene therapy still poses many challenges for use as a routine medical practice.
In germ line gene therapy, germ cells can be altered by the insertion of functional genes that integrate into their genomes, thus being inherited by later generations. However, numerous ethical, religious and technical reasons restrict this for application in human beings. Conversely, in somatic gene therapy, the genes are introduced into somatic cells; therefore the effects will be restricted to the individual and will not be inherited by the offspring. However, we must be especially cautious with the vectors used for the gene delivery, particularly when viral vectors are used. Concerns about the risks of AVVs were raised during a gene therapy clinical trial in 1999, after an 18-year-old participant, Jesse Gelsinger, died because of a massive immune response triggered by the viral vector.
In our view, gene therapy represents a very promising tool that may ameliorate many human genetic diseases and neuropathological conditions accompanied by protein dysregulation at the expression and/or functional levels in the future. However, the nature of the diseases themselves and their genetic links must be more deeply understood before applying gene therapy. In the same way, the biological consequences of gene therapy need further evaluation, including its short-range and long-term side effects. Nonetheless, although gene therapy is still in its infancy, it has already been used in vivo with some success. The recent findings related to the treatment of acquired motor neuropathies described in this chapter highlight the therapeutic potential of this novel genetic tool.
Viral vectors were developed and real-time imaging experiments were performed in the Dr. Sergey Kasparov’s laboratory (University of Bristol, UK). This work was supported by grants from the Ministerio de Ciencia e Innovación, (SAF2008-01415) and the Consejería de Innovación, Ciencia y Empresa from the Junta de Andalucía, (PAI2007-CTS-02606), both co-financed with FEDER funds and the Mútua Madrileña Foundation, Spain. We thank Elaine Lilly, Ph.D. (Writer’s First Aid), for English language revision of the manuscript.
Derived from the Ancient Greek word ‘trauma’ (=wound) and preserved in its etymological originality, psychological trauma is a phenomenon that involves an injury to the psychological matter. Trauma is generally defined as any experience that is felt to be unbearable that shatters the human psychic potential and affects the human capacity to relate, and feel kinship with others authentically [1]. According to Kalsched, trauma refers to a type of psychological injury to the capacity to feel, which occurs when we are given more to experience than we can consciously bear, especially if we the lack resources to metabolise the mental states that emerge. Such an experience may disturb our sense of inhabiting the world in a coherent, safe, and meaningful manner [2]. As Greening quotes: when we experience trauma, our relationship with existence itself is shattered [3].
\nTraumatic experiences are broadly associated with a painful life event, which is characterised by its intensity, by the difficulty of the person to respond adequately to its sequalae and by its pathological long-lasting effects on the psychic organisation [4]. Thus, psychological trauma is the unique individual experience of a single event or enduring conditions, in which: (a) the individual’s ability to integrate their affective experience is overwhelmed, and (b) the individual subjectively experiences a threat to life, psychosomatic integrity, or mental sanity [5]. The individual may be left feeling emotionally, cognitively, and physically overwhelmed, while common comorbid diagnoses associated with traumatic experiences include post-traumatic stress disorder, mood disorders, anxiety disorders, substance misuse, eating disorders and personality disorders.
\nThe sequela of trauma commonly involves a sense of current threat, betrayal of trust, violation of psychological and somatic boundaries, loss of power, entrapment, helplessness, confusion, pain, dissociation and loss [5]. Broad examples of events that are associated with a traumatic sequela involve relatively impersonal events like natural disasters and accidents, or events of a personal character like many forms of abuse including psychological, sexual and physical assaults, wars and torture. Additionally, events of commission like interpersonal violation or events of omission like neglect and abandonment, which are not necessarily socially constructed as traumatic, may still result in the individual experiencing a sense of threat to their integrity.
\nThe very fact that traumatisation is predominantly an esoteric, idiosyncratic experience renders notions of objectivity somewhat difficult to infer. For this reason, central to the formulation of traumatisation is an appreciation of the uniqueness of the individual’s subjective lifeworld [6] and the conditions that may have been shattered as a result of exposure to psychologically wounding experiences. It is the subjective experience of the objective events that constitutes the trauma…The more you believe you are endangered, the more traumatized you will be…Psychologically, the bottom line of trauma is overwhelming emotion and a feeling of utter helplessness. There may or may not be bodily injury, but psychological trauma is coupled with physiological upheaval that plays a leading role in the long-range effects (Allen [6], p. 14).
\nAt a neurobiological level, many studies have shown that the effects of environmental stress on the brain are being mediated through molecular and cellular mechanisms. Neuroimaging research findings found permanent structural changes in the prefrontal/frontal lobe volumes of the brain, as well as alterations in neurotransmitter systems in chronically maltreated children [7]. Additionally, rapid increase of dopamine under discrete or prolonged traumatic stress has been shown to cause DNA mutations in brain tissue. The main implication from these findings is that early repeated trauma may lead to permanent brain changes associated with psychopathology such as mood disorders. Even more crucially, Schore has highlighted the effects of relational trauma in the developing brain of the infant, by showing that early experiences of deprivation and neglect in the attachment system can result in an impairment of the limbic system and critical neuronal cell death, associated with future aggressive behaviour and affective dysregulation [8].
\nOver the years, a plethora of theoretical approaches and research studies examined the immediate and long-term psychosocial consequences of intensely traumatic events, and several psychological models have attempted to conceptualise and treat clinical presentations arising from traumatic experiences. Although psychotherapies began with traditional analytic approaches, other schools of therapy have examined post-traumatic syndromes like PTSD, including cognitive behavioural therapy (CBT), existential and humanistic counselling, dialectical behaviour therapy (DBT), and eye movement desensitisation and reprocessing (EMDR) therapy. Effective treatments for PTSD include trauma-focused cognitive-behavioural therapies, psychodynamic psychotherapy, existential-humanistic counselling, EMDR as well as integrative psychotherapies [9, 10, 11, 12, 13, 14]. With the focus of the current chapter on psychological trauma rather than on PTSD, the ensuing discussion will address theoretical contributions from developmentally focussed approaches residing mainly in the psychoanalytic/psychodynamic paradigm.
\nThe relationship between trauma and mental illness was initially investigated by the French neurologist Jean Martin Charcot, who was treating traumatised women presenting with what was known as hysteria at the time (Greek υστέρα = uterus) in the Salpetriere hospital [14]. Hysteria referred to as a set of symptoms including amnesia, paralysis, convulsions and sensory loss that would be traditionally treated with hysterectomy. Charcot observed that these symptoms may have had a psychological origin and noted that traumatic events could induce a hypnotic state in his patients and was the first clinician who captured the process of post-traumatic dissociation owing to the endurance of unbearable experiences. Charcot’s student, Pierre Janet, continued to examine the relationship between traumatic memories and dissociation. More specifically, Janet studied the impact of traumatic experiences on his patients’ behaviour and personality development. Janet observed an association between his patients’ intense affects and their recollections or interpretations of their traumatic experiences, and found that through hypnosis, abreaction and re-exposure to the traumatic memories, patients’ symptoms were alleviated [14, 15]. A substantial contribution to the early studies of trauma arose from the works of Freud and Breuer cited in [16], starting with the famous case of Anna O, who initially presented with symptoms of hysteria but was later conceptualised as a case of dissociated, repressed trauma originating in her relationship with her father. In their studies on hysteria in 1893, Freud and Breuer referred to traumatic dissociation as hypnoid hysteria and highlighted its relationship to a traumatic antecedent. Implementing hypnotic techniques as an initial form of treatment led to the gradual development of the psychoanalytic approach with a focus on free association, abreaction and interpretation of unconscious intrapsychic and relational processes, as central to the treatment of traumatic presentations like hysteria [14]. In working with soldiers after World War I, Freud observed that his patients often re-enacted their battle experiences and noted that traumatic dreams have the characteristic of repeatedly bringing the patient back into the situation of his accident. In 1941, Kardiner, another psychoanalyst working with U.S. veterans from World War I, also studied the aftermath of trauma and his observations resembled Freud’s and Ferenczi’s postulations on the nature of re-enactment, a construct referring to an unconscious tendency to re-experience traumatic scripts: the subject acts as if the original traumatic situation were still in existence and engages in protective devices which failed on the original occasion (Kardiner [17], p. 82).
\nThe enormous impact of the Vietnam War on the psychological well-being of soldiers inspired more organised studies on trauma [14], while a growing interest in traumatisation within civilian contexts, particularly abuse and domestic violence, eventually led to the development of a diagnostic classification of trauma-related syndromes known as post-traumatic stress disorder in the Diagnostic and Statistical Manual (DSM-III) [18].
\nPost-traumatic stress disorder was originally classified as an anxiety disorder in the DSM-III and DSM-IV and is characterised by aversive experiences of anxiety, maladaptive behaviours, somatic symptoms as well as physiological responses that arise and develop following an individual’s exposure to a psychologically traumatic event. Recent reviews in the diagnostic literature now classify PTSD within the trauma- and stressor-related disorders in the DSM-5. PTSD symptomatology as outlined in the diagnostic manuals is theorised to result in clinically significant distress or impairment in several aspects of life activity, like occupation, social relations and other major areas of everyday functioning [19].
\nAlthough the diagnosis of PTSD captures a set of symptoms related to post-traumatic syndromes, it does not address developmental causes and childhood antecedents, nor does it offer a more complex and comprehensive view of intrapsychic and psychosocial stressors that exert influence on personality development and trauma-related pathology or distress [14]. For this reason, trauma has been conceptualised as ontologically different to PTSD. According to McNally, naive realists posit PTSD as an objective, timeless, universal psychobiological entity emerging in response to extreme stressors, while social constructionists assert that it is a cultural artefact arising in the wake of the Vietnam War [20]. As Young quotes: The disorder is not timeless, nor does it possess an intrinsic unity. Rather, it is glued together by the practices, technologies, and narratives with which it is diagnosed, studied, treated, and represented and by the various interests, institutions, and moral arguments that mobilized these efforts and resources [21].
\nWhile trauma is always an antecedent to PTSD [20], the two are not synonymous and it is important to emphasise that PTSD does not capture post-traumatic manifestations in their entirety. While all PTSD sufferers will have experienced some form of injury at some point in their lives, not all traumatised individuals will go on to develop PTSD. Yehuda and McFarlane [22] have shown that psychological trauma does not necessarily lead to PTSD but may precipitate other symptoms and syndromes. The authors propose that factors not yet well understood determine the variability of individual responses to trauma. Numerous psychiatric diagnoses in addition to or other than PTSD have been identified in traumatised patients including depressive syndromes, anxiety disorders, dissociative disorders, borderline personality, and substance misuse [21]. Chertoff asserts that PTSD rarely occurs alone and suggests that a range of trauma-related psychological problems, not fully captured in the DSM-IV framework of PTSD, occur together, requiring a more comprehensive approach [23].
\nResponding to the epistemological limitations and challenges of PTSD syndromes, Herman [24] suggested that ‘complex PTSD’ should be included as a new diagnosis that would address the multiple origins of trauma and their impact on all aspects of a person’s life including personality disorders. According to Herman, the lack of an accurate diagnostic concept has serious consequences for treatment, because the connection between the client’s presenting symptoms and the traumatic experience is frequently lost. Attempts to fit patients into the mould of existing diagnostic constructs generally result, at best, in a partial understanding of the problem and a fragmented approach to treatment.
\nMore recently, Ford and Courtois developed a more comprehensive model of ‘complex trauma’, conceptualising it as the inability to self-regulate, self-organize, or draw upon relationships to regain self-integrity (Ford and Courtois [25], p. 17). Complex trauma is associated with histories of multiple traumatic stressors and exposure experiences, along with severe disturbances in primary caregiving relationships (Ford and Courtois [25], p. 18). Therefore, a comprehensive formulation of complex trauma calls for a treatment model that addresses the immediate post-traumatic symptoms, which are not always limited or even relevant to PTSD but may instead manifest themselves in other clinical presentations. Additionally, the diversity of traumatic ramifications is fundamentally observed within the realm of human relationships and characterological formation. Because of the far-reaching implications of complex trauma for an individual, psychodynamic psychotherapy has been positioned in the literature as more effective for complicated types of PTSD and the broader interpersonal sequelae of trauma. Clinical and empirical evidence suggests that psychodynamic approaches may result in improved self-esteem, enhanced ability to resolve reactions to trauma through improved reflective functioning, increased reliance on mature defenses with concomitant decreased reliance on immature defenses, the internalization of more secure working models of relationships, and better social functioning [26].
\nMuch of the trauma literature in psychology has been based on classic psychoanalytic formulations [14], and later revisions include contemporary psychodynamic approaches, arising from the school of object-relations and interpersonal psychoanalysis [14, 15, 16]. In psychodynamic theory, trauma is understood to have a shuttering effect on the corporeality of the individual that requires psychological adaptation and results in a distinctive yet polymerous psychological sequelae. However, in comparison with competing theories, the psychodynamic model is distinctive in its emphasis on developmental history, unconscious function, and interpersonal processes [16].
\nHealthy human development throughout childhood is conditioned upon safe and stable interpersonal and contextual experiences, which shape the individual’s intrapsychic dimensions and relational capacities. The interplay between the intrapsychic and the interpersonal determine to a large extent adult functioning and well-being, particularly in regards to one’s sense of self and experience of the world around them [14]. Traumatic experiences hold the unique characteristic of interfering with children’s natural developmental potential—that is, their ability to feel safe within their psychosomatic self and establish a sense of belonging within their human environment; to unequivocally experience mental states and authentically express them; to recognise others’ mental states and empathically respond to them too; to imagine and to symbolise; to act autonomously but rely on others too; and finally to be able to love and create.
\nFreud’s clinical observations led him to theorise that mental life is instinctually motivated by two oppositional drives—Eros and Thanatos. Eros literally translates into romantic love and Thanatos into death. For Freud, the interplay between the love instinct (also referred to as the life instinct) and the death instinct (aggression) is what produces intrapsychic conflict and results in anxiety and resultant pathology—For it is through all bad feelings and conflicts are known [27]. Freud chose his words with novelty. His reference to the life instinct as ‘Eros’ was not random as romantic love has been universally celebrated as the highest of all emotions. The idea that love is one of the most fundamental forces in the world, if not the most fundamental force, has a long and influential history [28]. Ancient Greek philosopher Empedocles argued that it was through love and strife that the four elements of nature—fire, water, air and earth—were bound together to create everything around us. Plato argued in the Symposium and the Phaedrus that love is our response to the forms—the higher form of reality, the model for everything that exists [28]. Plato defined love as the joy of the good, the wonder of the wise and the amazement of Gods. But for Freud, the concept of love went far beyond affective experience—it necessitated an impelling function that preserved all the good for personality and civilization alike. For Plato, it is love that leads us to the forms, the ground of reality. For Freud, anything that we seek to understand and relate to, we invest with libido—in other words, we love [28]. Despite Freud’s emphasis on the nature and function of the libido per se (the sexual drive and its symbolic expressions), he captured the importance of love both in intrapsychic and interpersonal terms predicating that love and reality are connected at the very deepest level [28].
\nFreud’s original seduction theory postulated actual sexual experiences during infancy and early childhood as the cause of all traumas and the basis for neurosis [16]. However, he lost faith in his ‘seduction hypothesis’ for a number of reasons: first, he came to doubt the prevalence of sexual abuse he encountered in his clinical practice, and second, during the course of his own self-analysis, his repressed sexual feelings towards his mother led him to acknowledge infantile sexuality as a driving force of personality development, from which he constructed the Oedipus complex. This revision led Freud to privilege the role of repressed unconscious fantasy and intrapsychic conflict over interpersonal factors in the development of traumatic neurosis [16]. Freud went on to distinguish between traumatic and anxiety neuroses on the basis of real occurrence versus unconscious fantasy and the resultant intrapsychic conflict (e.g., desire versus rage). According to Freud’s clinical evaluations, the pathogenic agency was invested in the memory of the trauma and the patients’ defences to tackle the emerging anxiety like denial, repression and dissociation. A discharged affect of the attached traumatic experience usually resulted in these memories transforming into tolerable ordinary recollections, accessible to the conscious mind. When a reaction discharge was, however, impossible, then these affectively undischarged memories were theorised to enter a second consciousness where they became secrets, either isolated from the conscious personality or available to it in a highly summarised form and often unconsciously acted out [29].
\nA related interpersonal observation was that of ‘traumatic re-enactment’—the human tendency to repeat earlier patterns of relating in an attempt to master the conflict arising from the repressed, traumatic experience. Freud captured this phenomenon in his early writings on ‘remembering, repeating and working through’ [30] and termed it compulsion to repeat (Freud [30], p. 151). Attending to his patients’ relational patterns using free association, Freud discovered the notion of transference, a relational phenomenon whereby the therapist (and other important people) symbolically represented a figure from the patient’s past. By closely attending to the manifestations of the transference (e.g., how the patient treats the therapist, their expectations, wishes and anticipated roles) and gradually interpreting its aim (e.g., a wish to dominate an abusive parent by overpowering the therapist), Freud was the first clinician who attributed therapeutic change to the processes of the therapeutic relationship. We must be prepared to find, therefore, that the patient yields to the compulsion to repeat, which now replaces the impulsion to remember, not only in his personal attitude to his doctor but also in every other activity and relationship, which may occupy his life at the time-if, for instance, he falls in love or undertakes a task or starts an enterprise during the treatment (Freud [30], p. 151).
\nIn addition to Freud’s attendance to intrapsychic conflict and fantasy, the role of external traumatic experiences has been widely highlighted in the writings of many classical psychoanalysts including Carl Jung, Sandor Ferenczi, Anna Freud and Alfred Adler, all of whom emphasise the reality of early childhood traumatic experiences and their impact on the formation of mental representations of self and other. Followers of Freud like Klein, Winnicott, Fairnbairn and Bion developed his original model further and included the realm of human object relations, which is the centrality of relationships, particularly attachment processes and infancy experiences, in the formation of personality and character pathology following trauma.
\nA significant departure from Freud’s conceptualisation of libido was offered by Ronald Fairbairn who believed that the libido was not purely pleasure seeking but object seeking, and as such, Eros could be understood as a drive for relational gratification [31]. When libidinal urges become thwarted in childhood, either via the frustration of the child’s dependency strivings or because the child’s efforts to establish affirming and safe interactions are not met in a reciprocal fashion by the care givers, the child turns away from external reality. In place of those connections, the child creates a fantasy world of internal objects that contains features of the real-world objects with whom the child cannot establish and maintain a meaningful relationship (Ringel and Blandell [14], p. 67).
\nDrawing on his work with abused children and schizoid presentations in adulthood, Fairbairn noted that traumatic experiences in infancy cause the developing child to feel unloved as a person in their own right and also to interpret their own love towards the caregivers as essentially bad, worthless or destructive. The child then absorbs the parental characteristics and identifies with the unresponsive features of the parents: isolated, depressed, masochistic, bullying and self-destructive [14]. Fairbairn asserted that by internalising these pathological character traits, the child re-establishes a connection to the parent, who is unavailable in other, healthier ways. This type of internalization of the parents also necessarily creates a split in the ego: part of the self remains directed toward the real parents in the external world, seeking actual responses from them; part of the self is redirected toward the illusory parents as internal objects to which it is bound (Mitchell and Black [31], p. 120) [14]. Traumatic re-enactments in subsequent relationships were thus associated with the painful sequelae of the object-seeking behaviour.
\nFerenczi’s contributions to the theory of trauma equally favour experiences of exogenous nature and their exerting influence on personality development. Ferenczi studied phenomena of regression, repetition and acting out in treatment with abused patients, and apart from his emphasis on dynamics of the real traumatic relationship between the child and the perpetrator, he highlighted another important traumatic phase: the denial of the traumatic event by significant people in the child’s life, most notably the child’s mother. For Ferenczi, the environmental rejection of the child’s living nightmare represented the most pathogenic component insofar as the child’s reality was inadvertently invalidated, thus enhancing dissociation and depersonalisation leading the child to resort to distorting their own reality in order to survive. The problem with this type of defence is of course what Freud always referred to as the return of the repressed in the form of neurotic symptoms [14, 16, 32].
\nFor Winnicott, the experience of trauma is central to the development of the ‘false self’, which comprises an inhibited, fragile and often hidden sense of identity, akin to an insecure attachment system, with an impoverished capacity for trust and authenticity [33]. In Winnicott words: I find it useful to divide the world of people into two classes. There are those who were never ‘let down’ as babies and who are to that extent candidates for the enjoyment of life and of living. There are also those who did suffer traumatic experiences of the kind that result from environmental let down, and who must carry with them all their lives the memories of the state they were in at moments of disaster. These are candidates for lives of storm and stress and perhaps illness(Winnicott [33], pp. 123–124).
\nWinnicott wrote that babies enter life with an ‘inherited potential’ for a ‘true self’ that reflects their existential essence [34]. In ‘the holding environment’ provided by an available, containing, responsive and emotionally attuned maternal figure, the baby’s authentic, spontaneous expressions originating from the Id, develop and their sense of identity becomes firmly established. Infants, on the other hand, who are exposed to repeated deprivations or impingements, do survive but at the cost of ‘living falsely’.
\nFor Winnicott, the false self is a necessary facade that the child erects to secure the mother’s love by being compliant with her inadequate adaptations or unconscious expectations. For example, a depressed mother might prematurely force an infant to be ‘cheerful’ and ‘strong’ by projecting all of her unconscious wishes for rescuing onto them; a child of very angry, unstable parents might be terrified from expressing any of its own darker emotions or a child of intrusive parents might be prevented from developing a capacity to be alone and regulate their emotions.
\nWinnicott’s observations uniquely highlight the role of relational trauma in personality development, which is often subtle and invisible to ‘the bare eye’ as it does not necessarily encompass the drama of sexual or physical abuse and their visible scars, but instead runs within the psychic vein like a colourless poison.
\nKohut’s self-psychology framework encompasses a novel model for understanding normative human development and developmental deviations, as well as a theory of psychopathology. At the centre of Kohut’s theory is the idea that healthy personality development presupposes the satisfaction of core narcissistic needs within the attachment system, such as the need for mirroring (building a coherent sense of identity), idealisation (establishing self-esteem) and twinship (fostering a sense of belonging) [35]. Kohut viewed psychological disturbance as both originating and resulting in deficient self-functioning, manifesting in a variety of forms (e.g., borderline conditions, pathological narcissism and other personality disorders as well as depression, anxiety disorders and sexual perversions). Kohut formulated these conditions as the result of chronically occurring, traumatic breaches in parental bonds, most notably in empathic failures [14]. Unlike many object-relation theories in which maturity is equated with separation and individuation, self-psychology sees the developmental line of self-object relations as extending from birth to death [14]. Thus, self-psychology is predominantly an interpersonal approach that addresses intrapsychic development on the basis of how core narcissistic needs are being met within the attachment system.
\nThe origins of attachment theory are found in Bowlby’s idea that the relationship of close proximity between the infant and the primary caregiver serves not only as a survival mechanism, but it also allows the infant to develop socially and emotionally. In other words, the close proximity to protective caregivers keeps the child safe during threatening times, while psychologically it creates a sense of ‘secure base’, allowing the child to explore the environment. Influenced by object relations, Bowlby developed the concept of internal working models (IWM) to describe the representational models of self and others stemming from the quality of early bonding experiences. Traumatic attachments characterised by experiences of abandonment, physical or sexual abuse, neglect or parental indifference are theorised to hinder various domains of psychosocial development, such as romantic and peer relationships, disturb affect regulation, and damage the self-concept [14, 16]. Contemporary psychoanalysts, Fonagy and Target, introduced the term ‘mentalisation’ to describe one’s ability to comprehend mental states in self and others (e.g., thoughts, feelings, intentions, and wishes) and developed a model that linked insufficient mentalising capacity to a pathological self-originating, in traumatic attachment, experiences [36].
\nThis section will present a case study summarising elements of psychodynamic treatment, process and outcome with a particularly challenging clinical presentation of substance misuse following developmental trauma. To preserve confidentiality but to maintain originality of the essence of treatment, all identifying details have been replaced with fictitious information. I borrowed my title from a novel written by Maro Vamvounaki in 2008 The phantom of the unspent love, which uniquely highlights the intricacies of inner deprivation and psychic unfulfilment, in an era where satisfaction is pursued at all costs. As the novelist highlights: Love is indeed the great completion of the existence, but only when it’s about the love that you give.
\nOlivia was a 48-year-old consultant cardiologist who sought therapy following years of opiate dependency syndrome and episodes of recurrent depression. Olivia’s distinguishing talent and formidable skill led her to a nomination to specialise as a heart surgeon, but due to her concealed heroin addiction, she ‘settled’ with cardiology alone (mainly teaching coupled with part-time consulting), acknowledging the potential risk involved were she to perform open heart surgeries. Olivia developed an addiction to smoking heroin (but never injecting) during her medical training and saw it as a way of forming peer relations and getting a sense of belonging when she was studying. Prior to smoking heroin, as a child, Olivia used to enjoy inhaling white spirit and reported doing so since the age of 9, when she started attending painting classes and spirit was largely available for art purposes. Her bizarre inhalant addiction went unnoticed by her immediate environment, and in the same fashion, Olivia kept her ongoing heroin abuse hidden, by controlling withdrawal symptoms before they would kick in. Olivia took substantial, intermittent breaks from her substance misuse that allowed her to maintain a reasonable level of professional and social functioning, but she eventually relapsed into her original habits, getting stuck into the vicious cycle of withdrawal and submission into cravings.
\nOlivia described a ‘normal’ childhood with plenty of material goods and toys, albeit dominated by parental conflict and fought relations within her family. She had some fond recollections of her mother as an affectionate and emotionally attuned figure but also described her as highly critical and controlling, often intruding into Olivia’s space leaving her feeling anxious and hypervigilant. Her father, on the other hand, was described as a remote and somewhat mysterious figure, whose main interest was his business and professional success. Her father was also described as the main source of stability in the home, and Olivia admired him for his physical appearance, wealth, moral values, and for offering them a comfortable, middle-class upbringing. Olivia had very little recollections from her childhood overall and the father in particular, as after the age of 9, he started travelling abroad a lot and so his presence was not felt in their home but was not missed either.
\nHer mother portrayed the father as cold and indifferent and frequently accused him of cheating on her (without evidence), causing her to feel neglected and chronically depressed. Olivia would often console her mother’s depression by keeping her entertained and by minimising the impact of her own neediness on her, by withdrawing or presenting content, despite feeling otherwise. Both parents held high aspirations from Olivia, and they hoped that she would study engineering in order to take over the family business in land development. While Olivia complied with their wishes and strived to please them, she chose a medical career instead, as she could not stand the thought of inheriting the family traditions.
\nOlivia also reported having a half-sister (from her father’s side) 10 years her senior, who died of an epileptic seizure when Olivia was only 6 and once again she reported very little memories from her or the period following her death. In fact, Olivia emphasised that the only connection she had with her half-sister was through a family album and stories she heard about her, without any real affective or mnemonic account of her own. Her half-sister did not live in the family home and this is how Olivia explained the gap in her memory.
\nAs an adult, Olivia consumed herself in short-term romantic relationships with ‘handsome and high-status’ men, but struggled to form intimate bonds reporting a feeling of suffocation and an aversion to co-dependency. She never married or had children, lived on her own and was fully dedicated to her career. Despite her professional success, social recognition and physical attractiveness, Olivia reported a chronic sense of emptiness, prolonged melancholia that was only interrupted by long-haul vocational activities, and a bizarre sense that she was fundamentally unloved. While she enjoyed conquering and dominating her lovers sexually, she reported episodes of anorgasmia (which she would also hide well in an attempt to get her lovers’ complete surrender to her sexual charisma and achieve a narcissistic triumph).
\nMore recently, she disclosed that she always had moments of silent crying during sexual intercourse that she would master by ending these relationships before they would develop into something more meaningful. Olivia sought therapy mainly to target her substance misuse as she became acutely aware that the use of heroin was serving a self-preservatory purpose that instinctively began to feel at odds with the causes of her existence. Olivia always maintained that she never suffered any trauma or abuse as a child, and became curious herself about the psychological origins of her long-term battle with addiction.
\nWhile Olivia struggled with her immediate recollection of her childhood memories, the material that we held was enough to explore the function of heroin as an object relation that would perform the soothing and self-regulatory functions the internalised object failed to successfully perform [33, 34]. Attending to her insecure attachment style and her relationship with her mother in particular, led us to identify Olivia’s unmet narcissistic needs as a child, in terms of self-object experiences [35] and how heroin would offer her the emotional tranquillity that she was so desperate for, but unable to provide to herself as an adult, in the same way the internalised mother failed to meet her child ego’s empathic needs.
\nDavies and Frawley assert that the traumatised child’s loss of a secure base may constitute the most pernicious and damaging psychological trauma. Because clients’ internal worlds contain partial or no representations of loving, protective objects, they never fully develop the capacity for self-soothing and self-calming at times of distress or for containment of the anxious states, disorganisation, and intense hyperarousal [37]. Relatedly, growing up with a depressed mother, Olivia’s main endeavour was to save her from her demonic occupations assuming the role of the hero (often successfully as that kept her mother going) but at the expense of establishing a coherent sense of self with an idealised internal other. Not being able to idealise a depressed and miserable mother, Olivia had no hero for herself and heroine by virtue of name and quality, symbolically satisfied that unmet narcissistic need for her [35].
\nAttending to this transference dynamic and interpreting the re-enactment within our relationship were paramount to the healing process. Olivia would often become acutely aware of any signs of vulnerability in me, and her immediate reaction would often involve some type of kind offer to enquire into my own shortcomings (Much of those offers were products of the transference in fantasy, in the absence of any real vulnerability on my part.) I often hypothesised whether she initially aspired to become a heart surgeon because she wanted to fix the mother’s heart and gain the love she was so desperate for. Olivia related to this interpretation well, noting the unconscious motivation behind the choice of her medical speciality, but she equally reported feeling loved by the mother despite her somewhat traumatic attachment to her. Rescue fantasies often occur as a result of parentification in childhood and are frequently repeated in adult relations where individuals enact the role of the hero in an attempt to achieve a narcissistic triumph and maintain the conditions of love they were initially subjected to, during childhood [35]. Rescue fantasies, however, also serve to unconsciously disavow the intolerable vulnerability of the wounded child’s ego state and aspire to create a dynamic where the rescued object is finally well enough to serve their own needs (representing the weak parent in the transference).
\nWinnicott captured the tremendous essence of growing up with a depressed mother in his poem ‘The tree’, where he illustrated the intricacies of the development of the false self. Indeed, Olivia struggled with her true self expressions in all facades of her adult life and emphasised how she would receive a sense of narcissistic gratification by being admired at work, saving her patients and being desired by men, but she never managed to feel loved for who she truly was. The therapeutic space acted as a new maternal, holding environment, and during the process, we utilised Kohut’s ideas of empathic resonance and attunement as the therapist’s primary mode of listening, and a focus on affect as an essential component of Olivia’s internal experience, which gradually aided her to build a more coherent sense of self that strengthened her capacity not only to mentalise but also tolerate her own emotional states (previously ‘anesthetised’ by heroin). This was accompanied by a reduction in the use of heroin and a stable compliance with her methadone schedule.
\nBy the second year of treatment, Olivia had already managed to maintain a stable drug-free regime and demonstrated a better capacity to regulate her affective experiences, despite relying on methadone and occasionally on legal anxiolytics. However, the relinquishment of her dependency from the drug unveiled Olivia’s core traumatic depression, and while her feelings of emptiness lessened, her sense of reported ‘unlovability’ became so raw that drove Olivia to form even more intense and rough sexual encounters whose only aim was pure libidinal gratification. It was almost as if an invisible force compelled her to seek immediate and unnegotiable physical proximity. But while she would report momentary narcissistic satisfaction from her lovers’ surrender to her physical tricks, all attempts at intimacy from prospective partners were met with what she termed ‘terror’ and an ensuing frantic escape into a schizoid retreat. In ‘a moment of meeting’ [14] with Olivia, I captured her desperation inside of me in the countertransference, which I only released when I declared the annulment of all of my previous interpretations regarding the unconscious motivation behind her intense sexual activity in the absence of attachment. Olivia was not seeking to receive love per se to satisfy a deficit in erotic resources, Olivia was seeking to grant love driven by an erotic surplus, in an attempt to re-unite with the lost object and secure her existence.
\nLove has been at the heart of philosophy, psychology and psychoanalysis since conception, but sadly often trivialised by contemporaries, due to the emphasis either on the individual’s libidinal urges as core motivators of mental life, or relational drives to secure survival. However, a distinguishing feature of the psychoanalytic approach is the awareness of the link between adult love and love in infancy [29].
\nFreud, cited in [29] postulated two models of libidinal relation—the initial narcissistic love of the ego (e.g., the mirror image sought in the other) and the anaclitic love of the object (e.g., the image of a parent). It could be argued that both types of love are anaclitic in nature as the individual requires mnemonic access to earlier patterns of relating to self and others for the repetition to occur, either pre-consciously or unconsciously. In 1912, Freud referred to love as an affectionate current akin to long-term attachment and differentiated it from sexual desire, which he referred to as a sexual current—both referring to a propelling, dynamic energy in the psyche. According to Freud, the split between affection and desire is experienced unconsciously in order to defend against punitive parental introjects, which are awakened by the experience of a new desired object in a sexually similar way as the subject felt towards the desired parent in childhood [39]. Freud referred to this as the mother-whore complex in male love, to illustrate the inability to maintain sexual arousal within a committed, loving relationship. This is, however, true for all relations. Clients with this complex desire a sexual partner who has been degraded, while they cannot desire the respected partner: where such men love they have no desire and where they desire they cannot love [38]. The person then develops two specific self-protective unconscious defences:
The first is an ascetic attitude towards desired objects in which disgust and humiliation are subliminally employed to thwart self-expansive, romantic urges.
The second is a moral masochistic reaction of guilt in the wake of desire. The person unwittingly assumes that yearning for new objects of desire is an act of disloyalty to parental introjects. To avoid this primal sin, the person seeks to evoke rejecting responses from the desired object by pushing them away. Interpersonal hurt then disrupts the transfer of incestuous impulses from a parental introject onto the new object. According to Freud, to defend against additional infantile patterns—repressive, seductive, magical longings for the ‘lost’ object of the parent–child bonding—moral masochism and asceticism work together, alternating in dominance [38].
In Olivia’s case, however, the process of erotic connection with the potential of leading to deeper intimacy was evoked by the thwarting of her own loving urges—not her partners’ evoked rejection, who on the contrary demonstrated an eager and often affectionate attitude towards her. In fact, Olivia’s attraction would immediately cease once the desired lover would attempt to transform into an affectionate partner.
\nSecondly, Olivia’s comfort with sexual proximity was anything but ascetic. Her compulsive hyperarousal, in fact, served to enhance the split between affection and desire in as much as desire for anonymous objects (short-term flings) would compensate for the lack of affection for an eponymous subject (long-term, stable attachments). The wake of the potential of affection towards an admirable and respected partner seemed to overwhelm her ego, sending her into a one-way schizoid retreat. Fairbairn observed that the schizoid’s main conflict lied in the individual’s perception of their own love as ‘bad’ and Olivia’s moral masochism manifested in the halting of her own expressions of affection towards the subject, not the other way round.
\nThe next logical question was ‘why’. There was nothing in Olivia’s narrative that would point to an earlier oedipal, traumatic experience that would account for the internalisation of her own love as destructive. I had already attempted to make a link between her father’s absence and her avoidant attachment style, which could have in part explained her distaste of bonds, but Olivia did not react with any substantial insight into that either. Having no material to work with from her earlier history and being confronted with Olivia’s severe depression, left me with no choice but to attend to her current acting out as the last resort to make sense of her suffering. Olivia continued to report outbursts of silent crying during sexual intercourse and affirmed her feelings of ‘terror’ every time a prospective lover invited her to reciprocate love in the form of relational affection—that is, every time the erotic physical yearning threatened to become an erotic relation.
\nDuring the later stages of treatment and while following this line of enquiry, Olivia met an older man who fell in love with her and resisted her continuous rejections by persevering into creating a long-term relationship with her. Olivia’s depression worsened over the coming weeks and she eventually relapsed into heroin, and suffered a myocardial infraction. Her heart was about to stop and she would no longer be confronted with the pain of needing to give love—the ghost of her unspent love would be long gone.
\nOlivia suffered a heart attack on the grounds of a hospital a few minutes before she was due to start work and was found immediately by a fellow doctor who proceeded to transfer her to the emergency room for resuscitation and emergency angioplasty. When she returned to therapy 6 weeks later, Olivia reported the following experience:
\nFollowing the coronary angioplasty and during the process of arousal from the general anaesthesia, Olivia entered a semi-conscious state where she began to recount memories from her childhood. This continued throughout her recovery process in the coming weeks where she was bombarded with lucid images from her past. Olivia recalled herself as a child visiting her half-sister’s house with her father, where she would be left to play on her own in the garden, while he would spend time indoors with the half-sister. Olivia recalled herself looking through the glass door into the living room and seeing images of her half-sister who was a teenager at the time, entering vague sexual encounters with the father. At the time, none of the scattered nude images or their proximal physical postures arising from gaps in the curtains made sense to her but evoked a strong feeling of terror. Olivia also remembered hearing her half-sister declaring her love for him on numerous other occasions and him demonstrating a bizarre, adoring attachment towards her. Olivia remembered attempting to ask her father about physical love between people and being heavily reprimanded for making such unacceptable enquiries. Olivia remembered feeling scared but forming a clear understanding at the age of 6, of what Eros meant, and what falling in love feels like. Olivia remembered sensing a confusing arousal inside her 6-year-old body—a precocious maturity. Olivia remembered telling her mother that she was scared of the father and her mother dismissing her. Olivia remembered that her half-sister died suddenly of an epileptic fit and that her father entered an inconsolable depression after that. Olivia remembered her father travelling abroad all the time following the death and being completely absent from her life. During his inconsistent returns home, Olivia remembered her father visiting her bedroom after midnight and her whole body shaking with terror, freezing into the foetal position. Olivia did not remember any sexual encounter with the father she admired, respected and loved, but remembered picturing the full meaning of love:death. Olivia painted many pictures of lovers that die in lust, at her after-school painting club, which she would often erase using white spirit.
\nIn the proceeding sessions, it was made clear that Olivia’s trauma had been dissociated from her conscious mind but fully absorbed and acted out in child play, in her behaviour towards adult relations, in her belief system and in her attachment style. Ferenczi illustrated how sexually traumatised clients may have little if any memory of the event largely due to the disbelief, minimization, and denial exhibited by adults in the child’s environment, whenever efforts were made to introduce the topic of seduction. In tandem with the child’s desire to maintain some sort of loving connection with the parents, these reactions are further reason for the child to disbelieve their own veridical recollections and to conclude that the seduction imagined was simply a fantasy production [14]. However, the memory of the veritable seduction experience and the resultant trauma do remain, creating a fragmented personality, in a way that each split or division in the personality behaves as though it does not know of the existence of the others. According to Ringel and Blandell ([14], p. 45) what is especially remarkable about this portrayal is how well it resonates with contemporary psychological formulations of the processes and phenomena of dissociation, now universally recognized as a hallmark of the post-traumatic adaptation [14].
\nFor Olivia, the operation of dissociation primarily allowed her to tolerate the reality of the distressing event by splitting off highly incoherent or overwhelming thoughts, memories and feelings [29, 32, 37] associated with witnessing her half-sister’s incestuous relationship with the father. The task of therapy was to help her integrate these memories into her personality while increasing her capacity to tolerate and process the affects and interpretations associated with the traumatic experience. Ringel and Blandell [14] quote Davies and Frawley who noted that with no self-reflective observing ego to provide even the rudiments of containment, meaning and structure to the traumatic events, the child exists in a timeless, objectless and selfless nightmare of unending pain, isolation and ultimately, psychic dissolution (p. 75). According to the authors, it is not just the traumatic memories that become dissociated from other experiences but also the organization of mutually exclusive systems of self and object representations (p. 75) formed in relation to traumatic experiences. The therapeutic process, as a result, must achieve integration not only of the memories themselves but also of clients’ varying experiences of self in relation to their fragmented worlds of internal objects [14].
\nThe concept of enactment was necessary and instrumental in Olivia’s treatment outcome in as much as it offered us a platform to observe the unconscious repetition of her primary relational and intrapsychic conflict, which led to the eventual dissolution of the dissociation and her successful abreaction of the trauma. As mentioned above, a central conundrum of Olivia’s traumatic depression was the split between her sexual behaviour and her attachment system—a dichotomy between desire and affection. Olivia seemed to have internalised a very antithetical vision of her father both as an oedipal object of desire, and as an attachment figure of resourcefulness and potential security. Witnessing the sexual incest and the simultaneous enmeshing attachment between her half-sister and her father, which was eventually interrupted by her death, led Olivia to develop a series of fantasies associated with her own capacity to express sexual desire and loving affection. In the final stages of treatment, Olivia was able to articulate how love, both in the form of affection and desire, threatened to bring about some sort of disaster (i.e., psychical death) in the same fashion her half-sister died for entertaining both internal states. Moreover, her father’s subsequent distant attitude led her to fear the consequences of her own affectional loving urges (possibly by introjecting his own incestuous guilt) and the safest way to prevent the impending catastrophe was to keep desire and affection separate. However, her compulsive urges to enter intense sexual relations were a reminder of her own uninvested or unspent love, and her failure to achieve a satisfactory outcome in terms of developing a lasting relational bond was possibly the most alerting signal of the enormity of her dual conflict: the intrapsychic vs. the interpersonal. According to Eagle ([39], p. 221) One classic and primary expression of an inadequately resolved Oedipal conflict is a relative inability to integrate sexual and lustful feelings on the one hand and tender and loving feelings on the other, that is, to have both sets of feelings towards the same person. One consequence of such an unresolved conflict, which often brings people into treatment, is great difficulty in establishing and maintaining long-lasting intimate relationships.
\nThe relationship between sexuality and attachment is not fully understood yet but contemporary theorists have attempted to shed some light on it by presenting it mainly as a parallel and antagonistic process facilitated by two distinct subsystems [40, 41] or as intertwined co-occurring systems that require integration for patients who might have suffered trauma [42]. The latter seems to be a more plausible explanation of the processes of attachment and sexuality in trauma, as the complete separation of the two, admits to a number of limitations, which go beyond the scope of this chapter. Eagle [41] supported the view that attachment and sexuality are functionally separable systems and, in certain respects, operate in mutually antagonistic ways. He further proposed that the integration of attachment and sexuality is a developmental challenge that is met by different people with varying degrees of success depending, in part, upon their individual attachment pattern. With regard to Freud’s postulation of moral masochism stemming from incestuous longings, Eagle correctly argued that whereas an incest taboo is relevant to understanding the split between love and desire, there is little evidence that universal incestuous wishes play a central role in accounting for that phenomenon [41]. Holmes [39] noted that it is not uncommon for partners to be intensely attached to each other with a relative absence of sexual interest and conversely that sexual involvement may preclude an attachment relationship. Fonagy also remarked that that these systems are separate and at most loosely coupled [39]. Diamond emphasised this separation by noting that desire is governed by the sexual mating system (p. 174), the goal of which is reproduction, whereas love is governed by the attachment or pairbonding system…the goal of which is the maintenance of an enduring association (p. 174) for the purpose of survival of dependent offspring [43].
\nWhile these observations hold tremendous theoretical value, they do not account fully for the intertwining between the two, given that Eros does not necessarily aspire to reproduction, but to other forms of connection including intimacy and unity. The latter is especially true for same-sex desire where the innate reproduction code is intact, but does not drive sexual longing per se. Kirkpatrick pointed out that the need for intimacy seems to be greater among women than men in homo-erotic relationships, and that genital release may not be their primary organising and motivating factor.
\nMoreover, we do not hold sufficient research outcomes to validate the claim that some sexual relations do not involve an attachment relationship at all. It may well be the case that the attachment dynamic is dominated by distance and minimisation, in which case we might be dealing with a biphasic defensive operation of symbolic character, rather than a complete lack of connection between lovers. Sexual arousal is indeed heightened by unfamiliar, distant and mystic attractions, but this may not necessarily preclude the undercurrent of attachment—in fact, examining the phantasies of the object representation may have a lot to say about the nature of these psychosexual dynamics beyond pure instinctual organisation.
\nEagle [41] noted that with regard to the transference dynamic, the more unresolved one’s early attachment relationship, which is characteristic of insecure attachment, the more one will react to current partner as a stand-in for a parent, and the less able one will be to experience one’s current partner as a sexual figure. That one continues to be avoidant toward current partner strongly suggests that one is continuing to react defensively, as if one were experiencing current partner as rejecting and/or intrusive, similar to the way one experienced the early parental figure [41].
\nOlivia’s traumatic depression was reinforced by her dissociation, concealed by her substance misuse and further elaborated by her inability to invest her loving urges towards potential partners, because attachment was equated with death in fantasy. The same did not apply to her sexuality though, apart from her chronic anorgasmia, which was another symptom of her traumatic exposure.
\nDuring the final stages of treatment, we focused on creating a more coherent narrative of her traumatic past, developed a new template of object-relations where love would meet desire in a non-destructive manner characterised by integration of the two; while allowing her to build a more stable sense of self that inclined her to attempt something a bit more meaningful with her partner than raw sex.
\nDavies and Frawley [37] strongly endorse the therapeutic relationship for acting as a vehicle through, which ‘soothing, undoing and redoing’ of the traumatised client’s life must finally occur largely based on the therapist’s ‘willingness to know’ the person fully. The relational model composed of various psychoanalytic contributions emphasises the intersubjective dynamics between client and, important others as well as client and therapist, around interpersonal enactments stemming from the traumatic experience. Enactments are seen not only as essential but also as instrumental in helping clients process and make sense of the previously unconscious relational patterns that they find themselves in, their impact on self and others, and their perception and interpretation of others’ words and behaviours.
\nStudies of traumatised children have demonstrated unequivocally that infants and toddlers have the prerequisite emotional and cognitive capacities for trauma to have persisting effects on their psyche. Traumatised children can retain some type of internal representation of their trauma for years, as demonstrated through trauma-centric behavioural re-enactments, affective responses to traumatic triggers, expressive play, sensory and somatic symptoms, and even verbal recall [44].
\nHistorically, a conception that has had wide currency within the psychodynamic school of thought is that psychological trauma in childhood both interrupts development and scars it forever. The overwhelming of the ego induced by psychological trauma is theorised to exert enduring memory imprints that cause intrusive flashbacks, emotional re-experiencing, traumatically driven behavioural re-enactments, trauma-centric fears, and disturbing dreams [20, 21, 22, 23, 24, 25, 26, 42].
\nThe trauma is seen as having an enduring organisational influence on the client throughout the life span, a conception Gaensbauer and Jordan termed as a ‘full-fledged’ repetition. Analysing cases of traumatised adults and children, Herman ([24], p.32) concluded that long after the trauma is past, traumatized people relive the event as though it were continually recurring in the present…the traumatic moment becomes encoded…and breaks spontaneously into consciousness, both as flashbacks during waking states and as traumatic nightmares during sleep [24, 44]. Olivia’s traumatic depression lied in the early sexual trauma she was exposed to and although she did not have any recollections of being sexually abused herself, the enormity of the terror she witnessed damaged her developmental potential and the capacity to form healthy relationships driven by a constant battle between desire and affection towards an object that held oppositional qualities; a battle of gifting love driven by a lack of security. Lacan’s famous aphorism on love was loving is to give what one does not have. For Lacan, this is the essence of loving—the key to love, to being able to love, is to accept one’s lack: One cannot love except by becoming a non-haver, even if one has [45].
\nThe author declares no conflict of interest.
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",metaTitle:"What Does It Cost?",metaDescription:"Open Access publishing helps remove barriers and allows everyone to access valuable information, but article and book processing charges also exclude talented authors and editors who can’t afford to pay. The goal of our Women in Science program is to charge zero APCs, so none of our authors or editors have to pay for publication.",metaKeywords:null,canonicalURL:null,contentRaw:'[{"type":"htmlEditorComponent","content":"We are currently in the process of collecting sponsorship. If you have any ideas or would like to help sponsor this ambitious program, we’d love to hear from you. Contact Dr. Anke Beck at anke@intechopen.com.
\\n\\nAll of our IntechOpen sponsors are in good company! The research in past IntechOpen books and chapters have been funded by:
\\n\\nWe are currently in the process of collecting sponsorship. If you have any ideas or would like to help sponsor this ambitious program, we’d love to hear from you. Contact Dr. Anke Beck at anke@intechopen.com.
\n\nAll of our IntechOpen sponsors are in good company! The research in past IntechOpen books and chapters have been funded by:
\n\n