Classification of SCI severity
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Manning is a Principal Scientist (Rank Grade 9) in the Coasts & Oceans Group at HR Wallingford (UK) and has over 23 years of scientific research experience (in both industry and academia) examining natural turbulent flow dynamics, fine-grained sediment transport processes, and assessing how these interact, (including both field studies and controlled laboratory flume simulations). Andrew also lectures in Coastal & Shelf Physical Oceanography at the University of Plymouth (UK). Internationally, Andrew has been appointed Visiting / Guest / Adjunct Professor at five Universities (Hull, UK; Delaware, USA; Florida, USA; Stanford, USA; TU Delft, Netherlands), and is a highly published and world-renowned scientist in the field of depositional sedimentary flocculation processes. Andrew has contributed to more than 100 peer-reviewed publications in marine science, of which more than 60 have been published in international scientific journals, plus over 180 articles in refereed international conference proceedings, and currently has an H-index of 24. He supervises graduates, postgraduates and doctoral students focusing on a range of research topics in marine science. 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There is no cure for SCI although recent advances in acute care interventions (e.g., removal of bone fragments, decompression, anti-inflammatory drugs) have increased survival and reduced neurological dysfunctions (Baptiste & Fehlings, 2007). Accordingly with the American Spinal Injury Association (ASIA) guideline, tetraplegic (cervical lesions) and paraplegic (thoracic lesions or below) patients are classified either as ASIA-A, ASIA-B, ASIA-C or ASIA-D (see Table 1). Quadriplegia also called tetraplegia is when a person has a SCI within the cervical area which results in paralysis of all four limbs. In addition to the arms and legs being paralyzed, the abdominal and chest muscles will also be affected which result in weakened breathing and the inability to properly cough and clear the chest. Paraplegia is when the level of injury occurs at the thoracic level or lower. Although they typically experience leg movement and abdomen problems, paraplegics can use their arms and hands.
ASIA-A | No voluntary motor control and no sensation below injury level |
ASIA-B | No voluntary motor control, some sensations below injury level |
ASIA-C | Some motor control (< grade 3) and some sensations below injury level |
ASIA-D | Some motor control ("/> grade 3) and some sensations below injury level |
ASIA-E | Normal voluntary motor control and sensation below injury level |
Classification of SCI severity
Advanced rehabilitation ‘activity-based’ strategies such as body weight-supported treadmill training or BWSTT (leg movements generated passively by manual assistance from therapists) and functional electrical stimulation (FES)-biking are increasingly used especially with motor-incomplete (ASIA-C and ASIA-D) patients. Indeed, given that spared descending pathways exist and, thus, some voluntary motor control remains in these subclasses of patients, it becomes possible to further increase voluntary ambulation using BWSTT training (Dobkin et al., 2006; Hicks & Ginis, 2008). However, motor system, metabolic outcomes or health benefits associated with these approaches remain unclear (Hicks & Ginis, 2008; Duffell et al., 2009). In turn, chronic SCI patients classified as motor-complete (ASIA-A & ASIA-B) generally experience greater health problems often referred to as ‘secondary complications’ that are associated with significant changes of the motor, locomotor, skeletal, cardiovascular, circulatory and hematologic problems (Huang & DeVivo, 1990; Bauman, 1999; Riegger et al., 2009; Rouleau et al., 2010,2011; Spungen, 2003). No safe, effective and regulatory agency-approved treatments against these chronic problems exist yet.
In the last few years, great therapeutic hopes for motor-complete SCI patients (ASIA-A and ASIA-B) have emerged from physical activity-based studies performed in adult complete paraplegic cats showing that basic locomotor movements (i.e., hindlimb stepping) can be restored partially with regular treadmill training, weight support, passively generated movement and administration (i.t. or i.p.) of drugs such as clonidine, an alpha-2 noradrenergic agonist (Barbeau et al., 1993; Chau et al., 1998). Regular assisted training combined with clonidine and a few other monoaminergic drugs have even induced, in some cases, episodes of overground walking with Canadian crutches in previously wheelchair-bound SCI patients (Barbeau et al., 1998). Clear evidence suggests that clonidine can, in fact, facilitate walking through reflex-mediated actions by decreasing spinal reflexes and hence spasticity and clonus (Waindberg et al., 1990; Remy-Neris et al., 1999). Unfortunately, at doses used for locomotor enhancement in some paraplegic patients, clonidine was also found to induce severe side effects even if given i.t. (i.e., bradycardia, sedation, hypotension - pers.com. Dr. Hugues Barbeau). It had therefore become imperative to identify other pharmacological strategies and compounds that could safely and more specifically enhance locomotor function recovery or reduce motor system changes and problems in chronic and, if possible, in motor-complete SCI patients (i.e., for whom BWSTT or other comparable approach does not yield beneficial effects). The identification of therapeutic approaches aimed at reducing or preventing motor system alterations in SCI or comparable chronic conditions (e.g., burn patients, AIDS patient with cachexia, etc.) would benefit both patients and health care systems for which associated costs are significant (approximately $100,000 - 400,000 per year/patient, Table 2).
Initial hospitalization | $140,000 |
1st year paraplegics | $152,000 |
1st year tetraplegics | $417,000 |
Averaged life time paraplegics | $428,000 |
Averaged life time tetraplegics | $1,350,000 |
Costs of SCI in U.S. dollars (source:
Although, these therapeutic approaches may not be designed to repair or cure SCI, they would nonetheless contribute at preventing (in acutely injured patients), reducing or reversing (in chronic SCI patients) secondary complications associated with motor system changes and significantly reduced physical activity (see also section 2.6).
The motor system may be divided into several organs and structures. There is the central nervous system (CNS) that comprises the brain and the spinal cord. Its one hundred billion neurons are involved in motor and sensory functions (Kandel et al., 2000). The brain consists of the pyramidal and extrapyramidal system specifically associated with voluntary motor control. These brain structures constitute the main command centres that control voluntary muscular contraction. Most of their neuronal commands are sent to neurons and motoneurons located in the spinal cord where sensory motor integration and final motor commands sent to muscle are organized for proper induction of coordinated movements.
In contrast, locomotion and other rhythmic and partially involuntary motor behaviours are largely controlled by signals and neuronal commands generated in the brainstem and spinal cord. In fact, complex neuronal circuits located in these non-cortical areas of the CNS are known to be capable of generating motor functions even in absence of descending inputs from cortical areas and other brain regions (Guertin & Steuer, 2009; Guertin, 2010). In fact, locomotion, micturition, ejaculation, scratching, erection, and respiration are amongst the motor behaviours that are mainly controlled by spinal cord and brainstem circuits (see Fig.1).
Neuronal networks in the spinal cord that control, brain-independently, complex motor behaviours. Respiration (not shown here) is also largely controlled by non-cortical structures including the brainstem (e.g., Pre-Bötzinger complex).
Thus, the CNS controls either directly or indirectly the muscular systems. Although some types of muscles such as the cardiac and smooth muscles are considered controlled by the autonomic nervous system and hormones, the striated skeletal muscle system is directly controlled by the CNS. This is the main reason why after SCI, an immediate and irreversible loss of sensory and voluntary motor control is found. This said, increasing evidence suggests that functions controlled mainly by the spinal cord can nonetheless be elicited despite SCI using specific pharmacological or electrical approaches (see section 2.6).
In humans, the striated skeletal muscle system comprises approximately 650 muscles. It is formed by different fiber types and properties including slow-twitch fibers (type I) and relatively fast to very fast-twitch fibers (IIa, IIb and IIx)(Table 3). The main action of skeletal muscles in motor control is to allow movement execution. Almost all skeletal muscles either originate or insert on the skeleton. When a muscle moves a portion of the skeleton, that movement results into flexion, extension, adduction, abduction, etc. (Martini & Nath, 2011).
The human skeleton consists of both fused and individual bones supported by ligaments, tendons, muscles and cartilage. Among several functions, it primarily serves as a scaffold for movements controlled by the CNS and muscles as mentioned earlier. The biggest bone in the body is the femur which is also the main skeletal structure affected after chronic SCI, disuse or immobilization. Finally, energy and other metabolic processes involved in motor control and movements largely depend upon the integrity of the circulatory and hematologic systems – i.e., distribution of erythrocytes and oxygen to muscles.
Type I | Slow twitch, high fatigue resistant, high oxidative, low glycolytic |
Type IIa | Moderately fast twitch, fairly high fatigue resistant, high oxidative, high glycolytic |
Type IIx | Fast twitch, intermediate fatigue resistant, intermediate oxidative, high glycolytic |
Type IIb | Very fast twitch, low fatigue resistant, low oxidative, high glycolytic |
Muscle fiber types and main properties
All in all, the main components of the motor system described above are changed and altered specifically in patients with complete and motor-complete SCI as well as in patients suffering of chronic disuse and immobilization (burn patients, AIDS patients, some patients with cardiac or pulmonary problems)(Huang & DeVivo, 1990; Bauman, 1999; Riegger et al., 2009; Rouleau et al., 2010,2011; Spungen, 2003; Lainscak et al., 2007).
In brief, all experimental procedures were conducted in accordance with the Canadian Council on Animal Care guidelines. Mice were generally housed 4-5 animals per cage in a controlled-temperature environment (22 ± 3°C), maintained under a 12h light:dark cycle with free access to water and food. Before surgery, pre-operative care was provided 30 minutes prior to anesthesia. It included subcutaneous injections of 1.0 ml of lactate-Ringer’s solution, 0.1 mg/kg of buprenorphine, and 5 mg/kg of Baytril, an antibiotic. Initially, complete anesthesia was conducted using 2.5% isoflurane in a cage of induction. Anesthetized animals were then shaved dorsally (2-cm) from the mid-dorsal area to the neck. Then, each animal was maintained under complete anesthesia using a specially adapted facial mask delivering directly 2.5% isoflurane to the animal. The shaved area was cleaned with 70 % (v/v) isopropyl alcohol and, then with 10% (v/v) povidone-iodine solution whereas eyes are protected from dryness using ocular lubricant. The first skin incision was made using fine scissors over 2 cm along the midline from the mid-dorsal area to the neck. Fat tissues (interscapularis fat) were cut and removed to expose the high-thoracic segments. The latissimus dorsi fascia was cut bilaterally to expose the vertebral column between the 4th and 6th thoracic vertebrae. Curved forceps were then used to tightly hold that area of the vertebral column that was cleaned from fascia and muscles to improve the grip. Forceps were also used to gently lift that part of the vertebral column which, once bent upward, eased the transection of the intervertebral ligaments between the 9th and the 10th vertebrae. This last part was critical to offer an open access for insertion of extra fine microscissors between the 9th and 10th thoracic vertebrae for the complete transection of the spinal cord. Then, the inner vertebral walls were explored and entirely, but delicately, scraped three or four times with fine scissors tips in order to sever any small fibres which had not been previously cut. It is important to scrape carefully to avoid severing the intervertebral ligaments located ventrally (i.e., if severed, it may lead to a dislocation of the vertebral column and corresponding bleeding). Throughout the transection procedures, bleeding although minor was controlled by applying pressure with cotton tips. The interscapularis fat was carefully replaced and the opened skin area was closed using 3 or 4 Michel suture clips. Michel suture clips are generally faster to install and are normally associated with less infection problems than typical suture threads. This overall surgical procedure was conducted under aseptic conditions using only perfectly cleaned materials and surgical tools – materials were previously autoclaved and tools were continuously sterilized throughout the procedure using a portable quartz beads-sterilizer).
Once the surgical procedures completed, anesthesia was interrupted and mice were placed in a large cage equipped with a heating pad placed underneath. It is critically important to use only minimal heating intensity (35°C) to avoid rapid dehydration, heat shock and death during the recovery period. Generally, the animals recovered completely within 15 min although we normally left them on the heating pad overnight with free access to food and water. The recovery procedure was found to be critical to ensure a high percentage of survival post-surgery (typically around 95% if everything is performed as described). The next day, the animals were replaced in their initial cage with their initial cage mates in order to reduce potential aggressions and fights.
Spinal cord histology. Luxol blue and Cresyl violet staining of a longitudinal section of the spinal cord from a non-laminectomized spinal cord-transected mouse one week post-surgery.
Postoperative care, provided a few hours after surgery as well as every day for the next 4 days, included injections of lactate-Ringer’s solution (2 x 1 ml/day, s.c.), buprenorphine (2 x 0.1 mg/kg/day, s.c.), and Baytril (5 mg/kg/day). Bladders were also manually emptied twice a day until a spontaneous return of some micturition reflexes. For voiding, the bladder was gently squeezed between the thumb (side of the bladder) and two fingers (e.g., the index and one other finger placed the other side of the bladder). This maneuver requires time and experience. In male mice, it was specifically challenging since, in addition, penises have to be maintained against a paper towel throughout the maneuver to improve successful voiding (i.e., it appeared to contribute, perhaps via capillary action, to urine expulsion outside the urinary tract). The belly and sexual organ were cleaned daily using paper towels and chlorhexidine gluconate solution (0.05 % v/v) to prevent urinary infection. Normally, with these procedures, mice that survived the firsts 24 hours, remained relatively healthy for a long period of time (i.e., several months). Finally, Michel suture clips were removed after 10 or 14 days post-surgery. Cages were cleaned regularly (ideally, cages needed to be changed every 3 or 4 days) and mice were cleaned, as described above, on a daily basis to prevent urinary tract infection. All in all, once anesthetized, this surgical procedure took no longer than five minutes whereas another 5-10 minutes was typically required for animals to recover from anesthesia.
This approach led to complete paraplegia (Figs. 2 & 3) – an immediate and irreversible loss of sensory and voluntary motor control below injury level (low-thoracic level). Although, it is possible to maintain these animals relatively healthy for severaonmonths post-spinal transection, a number of neuronal, muscular, skeletal, vascular, and hematologic changes were rapidly displayed. A detailed characterization of these changes is presented in the following subsections.
Video images of a paraplegic mouse placed on a treadmill. A complete loss of hindlimb movement is encountered immediately following the spinal cord transection.
Nearly all SCI individuals experience a drastic loss of bone mineral content (up to 30% at the femoral level) leading to a marked increase of fracture incidence within one year after injury (Ragnarsson & Sell, 1981; Garland et al., 1992; Wilmet et al., 1995; Lazo et al., 2001; Sabo et al., 2001). Although, the basic mechanisms underlying osteoporosis in post-menopausal women have been extensively studied, those involved in chronic immobilization and disuse have received considerably less attention. In animal models of disuse, traditionally in rats, hindlimb immobilization has been found to induce a drastic and sudden loss of femoral bone tissue suggesting that different mechanisms may be involved in disuse vs. estrogen-deficiency/aging-related osteoporosis (Bagi & Miller, 1994). For instance, a 10-30% decrease of cancellous bone has been reported within only a few weeks in the ipsilateral femur of rats that had their hindlimbs immobilized with a cast or an elastic bandage (Ito et al., 1994; Ma et al., 1995; Mosekilde et al., 2000). Comparable changes have been found in other models of disuse such as in tail-suspended rats (Wronski et al., 1989). Some of these disuse-related changes are believed to be mediated by both an increase of osteoclastic bone resorption and a decrease of osteoblastic bone formation (Rantakokko et al., 1999). On the other hand, growing evidence suggests that several factors other than mechanical unloading per se can influence the combination of cellular and molecular mechanisms underlying disuse-related bone loss. For instance, in the case of disuse induced by a lesion of the sciatic nerve, the loss of bone tissue in rats is caused partly by a disruption of the neurogenic innervation of the bone marrow (Zeng et al., 1996). Moreover, differential tissue- and biomarker-specific changes have been reported in the tail-suspension vs. sciatic nerve lesion models (Hanson et al., 2005). In the case of microgravity, bone tissue changes have been attributed mainly to a marked decrease of osteblast formation in young adult rats (Matsumoto et al., 1998). Taken together, those data suggest that the combination of various factors specific to each model and condition of disuse may dictate, to some extent, the different sets of molecular mechanisms involved in demineralization and bone loss.
Here, we characterized some of the main structural and functional adaptive changes occurring specifically within a few weeks in adult spinal cord transected mice. In brief, within a few weeks post-transection, paraplegic mice were weighed, sacrificed and the femoral bones dissected and cleaned of soft tissue. The femurs were wrapped in saline-soaked gauze and frozen at -20 degrees C in sealed vials until testing. For histomorphometry, the left femoral bones were fixed with paraformaldehyde, decalcified, paraffin embedded and stained with acid fuchine using the Masson’s trichrome procedures. Histomorphometric analyses were performed with a NOVA Prime, Biioquant’s image analysis system (R&M Biometric, Nashville, TN) for primary bone morphometric parameters. Three bone slices at the metaphyseal level were analyzed. For densitometry, measurements were made with the rigth femoral bones of sham and paraplegic mice. Bone mineral content (BMC, g) from the femora of each animal was assessed using dual-energy X-ray absoptiometry (DEXA, model Piximus II, Lunar Corporation, Madison WI, for details, see Kolta S, De Vernejoul M.C. et al. 2003). Bone mineral density (BMD, g/cm2) was calculated as BMC divided by projected bone area. Each femur was scanned separately for whole bone analysis. For biomechanical assessment, on the day of testing, the femur was slowly (4 hours) tawed at room temperature. They were placed horizontally on the three-point bending device (MTS, Eden Prairie, MN). The mechanical resistance to failure was tested using a servo-controlled electromechanical system (Intron, Instron, Canton, MA). The crosshead speed for all tests was 10 mm/sec until the femur fractured. Displacement and load values were acquired at 100 Hz, recorded and stored on PC. Off-line data analyses were performed to calculate maximal strength (N), stiffness (slope of the linear part of the curve to failure, N/cm), and elasticity deformation (N). Bones were kept wet throughout testing and used for histomorphometrical testing (proximal end).
All histomorphometric measurements and analyses were made from the metaphyseal area of the left femora. The cancellous bone volume was found to decrease by 25.2% in paraplegic mice (within 1 month post-transection) compared with control (non-paraplegic). The average trabecular bone thickness was found to decrease by 10.65%. The thickness was initially of 25.55 micron in the control groups and of only 22.83 micron in the paraplegic group. The number of trabecular bone areas decreased rapidly also after injury. In the control group, the average trabecular number was 3.38 nbr/mm2 whereas in the paraplegic group, it decreased to only 2.89 nbr/mm2 representing a 14.50% decrease. On the other hand, the trabecular separation, defined as the space between trabecular bone areas, increased after injury. In fact, on average, the trabecular separation increased by 24.03% within 1 month post-SCI (Picard et al., 2008).
The bone mineral density (BMD) of the left femora measured by dual-energy X-ray absorptiometry (DEXA) significantly changed after injury. The BMD was just below 0.09 g/cm2 in control and of 0.0731 in paraplegic mice. Bone mineral content (BMC) also proportionally decreased after injury (see sections 2.6 and 2.7 for further details).
The maximum force in N required for the crosshead to fracture the right femora at the mid-diaphyseal level was decreased by 13% on average within a few weeks post-transection (Fig.4D). The stiffness in N/mm was also reduced after injury with average values of 57.23 and 51.08 in control and paraplegic groups, respectively, representing a 10.8% decrease (Fig.4B). The elastic force decreased also by approximately 15% in early spinal transected mice compared with control (Fig.4C).
It is well-documented in various rat models that the contractile properties of slow twitch muscles change into more fast-like muscles after chronic spinalization (Roy et al., 1991; Talmadge, 2000). Hindlimb extensor muscles such as soleus (SOL) typically exhibit extended atrophy (e.g., up to 50%) and type I to type II muscle fiber conversion following spinalization in rats (Krikorian et al., 1982; Lieber et al., 1986 a,b; Midrio et al., 1988; Talmadge et al., 1995). Contraction and relaxation times as well as maximal tetanic force (Po) and maximal twitch force (Pt) have also been found to be importantly decreased in rat SOL several months after spinalization (Davey et al., 1981; Talmadge et al., 2002).
Evidence from other models of inactivity and immobilization suggests that some of these changes, in fact, are induced very early after inactivity and reduced muscular activity and loading. For example, a 10% loss of body weight (Pierotti et al., 1990) accompanied by a 40-50% decrease of SOL mass, TPT and 1/2 RT (Frenette et al., 2002) and a rapid reduction in slow myofibril proteins (Thomason et al., 1987) have been reported after 1-2 weeks of hindlimb suspension in rats. Comparable results have been found within less than 2 weeks in rats after spinal cord isolation (i.e., de-afferented and spinalized, Grossman et al., 1998) or in microgravity (Fitts et al., 2001). In addition, a 40% reduction of SOL cross sectional area has been found only 10 days post-spinal cord transection in rats (Dupont-Versteegden et al., 1999). The possibility that other early changes may occur after spinal cord transection is largely unexplored.
Bone mechanical properties. Two-point bending test (A) revealed decreased femoral stiffness (B), elasticity (C) and maximal force (D) in untrained spinal transected mice (spinal, black) versus control (intact animals, white)(unpublished data).
Here, we characterized some of the earliest adaptations in gross anatomy and muscle properties at only 7 days following spinal cord transection in adult mice (Landry et al., 2004). In brief, whole body weight was measured daily during the first week post-spinalization. After dissection of SOL for functional tests in vitro (see section below), animals were sacrificed with pentobarbital overdose. Forelimbs and hindlimbs were surgically removed just below the shoulder and the hip joints respectively. Paws as well as all parts of the pectoral and back muscles attached to the forelimbs were removed. Tests included weight measurement of the left forelimb and hindlimb as well as of the right SOL. To further assess muscle atrophy, limbs were weighed in air and in water to measure volume changes. Volume was calculated as follows with a volumic mass of 0.998 for water at room temperature (22oC):
For measurement of contractile properties, we anesthetized animals with pentobarbital sodium (50 mg/kg). The right SOL was carefully dissected and incubated in fully oxygenated Krebs-Ringer bicarbonate buffer solution maintained at 25oC and supplemented with glucose (2 mg/ml). In vitro measurement of muscle contractile properties was performed as described elsewhere (Côté et al., 1997). In brief, one tendon was attached to a rigid support at the bottom of the bath, and the other end was connected to an isometric force transducer (Grass FT-03) through a stainless steel hook. An initial resting period of 15 min was allowed before testing. Muscles were carefully stretched to their optimal length, defined as the length at which maximal isometric twitch tension is produced. One single twitch contraction was elicited and the following measurements were obtained: maximum twitch tension (Pt), time-to-peak tension (TPT), and one-half relaxation time (1/2 RT). After measurement of twitch parameters, muscles were stimulated for 1 s at frequencies of 10, 20, 35, 50, 80, and 100 Hz to determine maximal tetanic tension (Po, N/cm2). The value used for muscle density was 1.062 g/cm (Koh & Brooks, 2001) and the ratio of fiber length to muscle length used was 0.71 (Brooks & Faulkner, 1988).
We reported that paraplegic mice at 7 days post-surgery encountered a drastic loss in body weight (Landry et al., 2004). On average, a 24% decrease in weight was found at 7 days post-spinalization. A similar loss was found in another group of paraplegic mice that received instead daily injection of lactate-Ringer’s solution (2 ml/day, s.c.) during the first week post-spinalization suggesting that dehydration did not contribute to weight loss.
The specific weight of individual body parts was also examined in paraplegic mice. In intact mice, the average weight of forelimbs and hindlimbs was 436 and 1239 mg respectively. At 7 days post-spinalization, hindlimb weight decreased by 28% compared to intact mice. Interestingly, a 21% reduction in the forelimbs of paraplegic mice was also observed during the same period of time. Relative to body weight, the loss observed in hindlimbs was greater than the one in forelimbs. Similar reductions in volume were found respectively in hindlimbs and forelimbs.
Regarding properties, for soleus mass displayed significantly lower values (-32%) in untrained paraplegic mice at 7 days post-spinalization compared with intact animals. A 33% decrease of Po was measured at 7 days post-spinalization. The absolute tension generated at different frequencies of stimulation showed mainly that SOL force was reduced in paraplegic mice compared to control at stimulation frequencies above 35 Hz. On the other hand, maximal tension was reached at lower stimulation frequencies for paraplegics compared to control.
Our data showed also in soleus a change toward faster-type properties in the first few days post-immobilization (transection).The surprising initial and rapid conversion to slower contractile properties at 7 days post-spinalization is further supported by changes found in contraction and relaxation times (TPT and 1/2 RT respectively). TPT became slower (i.e., increased time of contraction) by 21% at 7 days compared to control. Similar changes were observed with 1/2 RT which became slower (i.e., increased time of relaxation) by 48% at 7 days post-spinalisation.
As mentioned above, it is well-known that there is an important shift in fiber phenotype distribution a few weeks post-SCI even more so in soleus. Generally, slow fibers tend to change for a faster phenotype after 2 weeks post-spinal cord transection. After spinal cord transection, 50-55% of the slow type fibers showed important fiber type conversion, shifting to a hybrid isoform (faster phenotype) whereas fiber type conversion was not observed in another hindlimb muscle, EDL, often classified as a purely fast-twitch muscle (Table 4).
Fiber type % | Non-TX | TX untrained |
EDL type II | 98.7 ± 0.3 | 98.7 ± 0.6 |
EDL hybrid | 1.3 ± 0.3 | 1.3 ± 0.6 |
SOL type I | 54.6 ± 2.6 | 2.9 ± 1.5 |
SOL type II | 45.4 ± 2.6 | 46.5 ± 2.5 |
SOL hybrid | 0 ± 0 | 50.7 ± 3.3 |
Fiber type conversion in normal (non-TX) and untrained paraplegic (TX untrained)(unpublished data).
Among the cardiovascular and pulmonary problems associated with SCI, deep venous thrombosis (DVT) is one of the most serious complications in patients that survive to the accident. Indeed, DVT constitutes the third most common cause of death in SCI patients (Waring & Karunas, 1991; DeVivo, 1999) and, despite prophylaxic methods (e.g. anticoagulant administration), a significant proportion of SCI patients will develop a pulmonary embolism caused by DVT (Deep et al., 2001).
Complete paraplegic and tetraplegic individuals are particularly vulnerable given that spasticity, typically found in incomplete SCI patients, may decrease the risks of DVT formation (Green et al., 2003). Generally, DVT formation is attributed to a combination of factors including also venous stasis, venous injury, and hypercoagulability. In turn, these factors facilitate platelet, LDL-cholesterol, and leukocyte adhesion, procoagulant system activation, and hence, thrombin generation. Although, few animal models of DVT and/or pulmonary embolism exist (Frisbie, 2005), none have been developed to study these complications after SCI which may explain why the specific mechanisms of DVT formation in paralytics remain poorly understood.
Here, we characterized, in spinal cord transected (Tx) mice, some of the physiological changes occurring after SCI that could possibly contribute to DVT formation (Rouleau & Guertin, 2007; Rouleau et al., 2007). Specifically, we characterized also alterations of deep vein diameter in the hindlimbs of Tx mice because venous distensibility and capacity changes may participate to DVT formation (Miranda & Hassouna, 2000). We took advantage of this experimental model to measure with great precision (µm), using in vivo fluorescence confocal microscopy, changes in diameter of the femoral and saphenous veins. All tests were performed weekly during one month post-Tx since risks of DVT in patients have been reported to increase by several folds specifically during the first few weeks after SCI (DeVivo et al., 1999).
In brief, we put the tail on a heated cushion to dilate the tail vein 10 min before injection. Then 200 µl of 5 mg/ml fluorescein isothiocyanate-dextran (FD-40) (Sigma, St-Louis, MO) dilute in injectable endotoxin-free dPBS (Sigma), was injected intravenously into the tail vein. Animals were killed by CO2 asphyxiation around 10 min after injection. The skin was cut to access to the femoral and saphenous veins. Microscope observation and measurement were performed with an Olympus BX61WI confocal system and analysed with Fluoview 300 (Carsen group, Markhan, Canada).
For hematologic data, peripheral blood was collected at various times post-transection by cardiac puncture. Each blood sample was analyzed for platelet quantification with a CELL-DYN 3700® automatic blood cell analyzer (CD3700)(Abbott Laboratories, North Chicago, IL).
We found by measuring deep vein diameter using in vivo fluorescent confocal microscopy techniques that the femoral and saphenous veins drastically increased in size after SCI compared with intact mice. This is illustrated in Fig. 5 showing typical examples from a control (left panel) and from a paraplegic mouse at 3 week post-TX (right panel). We can clearly distinguish that the femoral vein drastically increased in size post-transection compared with control. In fact, average values calculated for the femoral vein revealed, for control animals, an average diameter of 319 µm augmented to 458 µm at 3 weeks post-surgery (Fig. 5). Comparable increases of saphenous vein diameter were found after spinal transection (338 µm in control vs 433 in paraplegics)
The hematologic data revealed mild anemia that occurs as early as at 7 days post-transection. Specifically, average counts of erythrocytes (10.11 x 1012 /L in control mice) decreased to values ranging from 9.91 to 9.54 x 1012 /L in paraplegic mice. Hemoglobin concentrations were decreased from 164.9 ± 2.8 g/L in controls to 153.3 g/L in paraplegic mice. Decreased hematocrit levels were also found in paraplegic mice (range from 0.46 ± 0.01 to 0.44 ± 0.01 L/L) compared with controls (0.48 ± 0.01 L/L, Fig. 1C). In turn, platelet counts remained unchanged after spinal transection with levels of 16.76 ± 0.80 x 1011 /L in controls and 17.73 ± 0.75 in paraplegic mice (Rouleau et al., 2007).
The Central Pattern Generator (CPG) for locomotion is a network of neurons located in the lumbar area of the spinal cord that is capable of producing the basic commands for stepping even when isolated from supraspinal and sensory inputs (Grillner & Zangger, 1979, see also Guertin, 2010). Early evidence of a CPG emerged a century ago from the pioneer work of Sherrington (1910) and Brown (1914). In the 70s, low-thoracic spinalized rabbits and cats were used to show that an endogenous release of 5-HT induced by 5-HTP can generate fictive locomotor-like rhythms in the spinal cord (recorded with electroneurograms) of acute spinal cord-transected animals (Viala & Buser, 1971) or increase extensor muscle activity in regularly treadmill-trained and sensory-stimulated spinal animals (Barbeau & Rossignol, 1990, 1991). A clear demonstration of its existence was provided in 1979 by Grillner who could induce, with L-DOPA, locomotor-like neural activity in the motor nerves of completely de-afferented, curarized, and spinal cord-transected cats (Grillner & Zangger, 1979). In rats, the CPG was found, with activity-dependent labeling (e.g., c-fos), to be located mainly in rostral segments of the lumbar spinal cord (Cina & Hochman, 2000). Comparable results were found in mice where CPG activity was found to originate from lumbar segments with critical elements in L1-L2 (Nishimaru et al., 2000).
In the 80s and 90s, in vitro isolated spinal cord preparations were extensively used to study the pharmacological control of CPG neurons at the system and cellular levels. Initially discovered in lampreys, bath application of n-methyl-d-asparate (NMDA) was found to induce rhythmic activity (recorded from ventral roots) that shared locomotor characteristics – called ‘fictive locomotion’. This provided evidence that even a perfectly isolated CPG can be activated with drugs. Then, neonatal rat and mouse spinal cord isolated preparations were developed and used also to study in vitro drug-induced CPG-mediated locomotor-like neurographic activity. These studies have essentially revealed that bath-applied combinations of drugs such as NMDA, 5-HT and DA can best induce robust fictive locomotor-like rhythms in the mammalian isolated spinal cord (Cazalets et al., 1992; Kjaerulff & Kiehn, 1994). Although, these studies have revealed that several families of drugs need to be combined for enhanced CPG activation, most of the compounds used in vitro were synthetic neurotransmitters (e.g. 5-HT and DA) which, unfortunately, do not constitute good candidates for drug treatments because of poor selectivity (e.g. activation of all receptor subtypes) and incapacity to cross the BBB.
In humans, evidence of a CPG was provided after showing that \'automatic\' (involuntary) stepping-like movements could be triggered spontaneously under certain conditions or by epidural stimulation at the L2 level in SCI patients confined to a bed (Dimitrijevic et al., 1998). Although a completely isolated CPG can produce locomotor rhythms, sensory inputs (i.e. muscle proprioception, vision, etc.) were found to provide useful feedback signals to the CPG that can re-enforce muscle contraction and adapt stepping to external disturbances (Rossignol & Dubuc, 1994). However, none of these studies have identified a full CPG-activating drug that can, upon systemic administration, potently elicit acutely powerful weight-bearing stepping in complete SCI animals with no other stimulation/assistance (e.g., non-therapetically relevant tail pinching or other sensory stimulation).
Changes post-spinal cord transection were also found in sublesionally-located neurons (below injury level). Since most of these changes were found in neurons located in upper lumbar segments of the spinal cord, they were postulated to correspond with changes in CPG neuron candidates. Immediate early genes (IEGs) constitute a large family of genes well-known as early regulators of cell growth, differentiation signals, learning and memory. We reported in low-thoracic spinal cord-transected mice, that IEGs such as c-fos and nor-1 expression respectively increased and decreased within a few days in the segments L1-L2, specifically in the dorsal horn and intermediate zone areas (Landry et al., 2006). Changes in the lumbar spinal cord of rostrally-transected animals were of special interest since some of these segments (e.g., L1-L2 in mice) were shown to contain critical central pattern generator (CPG) elements as mentioned earlier. Given that IEGs are better known for their role in CNS development and plasticity, spontaneous changes of IEG expression (i.e., specifically c-fos and nor-1) in L1-L2 segments may be considered as among the first sublesional cellular events associated with altered cellular functions and properties post-SCI. This said, some of these changes may be associated also with other phenomena than plasticity or reorganization of spinal motor and locomotor networks. For instance, c-fos and nor-1 were used as markers in experimental models of pain and transient global ischemia suggesting a role in several functions (see Landry et al., 2006a).
Other key elements including transmembranal receptors may be considered good candidates for plasticity and reorganization of motor and locomotor networks located sublesionally following a spinal cord-transection (and probably to some extent also after partial injuries). For instance, we found using in situ hybridization increased 5-HT1A mRNA levels in L1-L2 segments in 5-HT7-deficient mice compared with wild-types (Landry et al., 2006b). This was interpreted as evidence suggesting that even greater changes may occur post-trauma in absence of functionally closely-related genes. Results in mice revealed also increased 5-HT2A mRNA levels in lumbar segments (laminae VII, VIII, and IX) several days after a low-thoracic transection (Ung et al., 2009).
All in all, it is unclear how these changes of neuronal properties and gene expression below lesion level may affect functional recovery and, specifically, the development of approach designed to reactivate behaviours-generating neuronal networks (e.g., CPGs for locomotion, micturition, ejaculation, etc.). Nonetheless, it has been postulated by others that such changes may contribute to increase sublesional network excitability and, thus, may facilitate training-induced learning and rehabilitation.
Given that no cure exists yet to repair the spinal cord, an interesting avenue to prevent or reduce some of the motor system changes described in previous sections of this chapter may be to pharmacologically induce episodes of locomotion. To achieve this, an alternative strategy could be to develop a CPG-activating drug treatment that could temporarily re-activate this sublesional network in tetraplegic and most paraplegic subjects.
Experiments mainly conducted in my laboratory since 2004 have led to a better understanding of pharmacological CPG activation in vivo. In brief, we found in completely low-thoracic spinal cord-transected mice that a few subtypes of blood brain barrier (BBB) permeable molecules can elicit partial CPG-activating effects (i.e., locomotor-like movements or LMs that resemble crawling - successive flexions and extensions coordinated in both hindlimbs without weight bearing)(Guertin, 2004a; Landry & Guertin, 2004; Landry et al., 2006; Lapointe et al., 2009). We subsequently found that drug combinations with some of these compounds including dopaminergic and serotonergic compounds (e.g., DA precursors such as L-DOPA combined with a decarboxylase inhibitor such as carbidopa, and a 5-HT1A receptor agonist such as 8-OH-DPAT or buspirone, etc.), could elicit significantly greater CPG-activating effects including large amplitude LMs with some equilibrium, plantar foot placement and weight bearing capabilities (i.e., real stepping rather than crawling, Guertin 2004b; Lapointe & Guertin 2008; Guertin et al., 2010, Guertin et al., 2011)(Fig.6). As mentioned earlier, this idea that drug combinations can produce apparently full CPG-activating effects was also supported by comparable findings in in vitro isolated spinal cord preparations (better and more stable fictive locomotor neuronal activities in isolated spinal cords, e.g., Cazalets et al., 1992; Kjaerulff & Kiehn, 1994; Kiehn & Kjaerulff, 1996; Jiang et al., 1999; Whelan et al., 2000).
This identification of a potent CPG-activating tritherapy (Guertin et al., 2010) recently received support from a special NIH program (Rapid Access to Interventional Development program) to conduct some of the preclinical studies (toxicity and safety pharmacology in rats). It has been determined that a tri-therapy composed of L-DOPA, carbidopa and buspirone is safe and ideally suited for further development at the clinical level (i.e., each drug is already FDA approved for diseases other than SCI and no abnormal pharmacology or toxicology data was found) as a first-in-class CPG activating drug treatment candidate. However, although efficacy in early chronic SCI mice has recently been demonstrated (Guertin et al., 2010; Guertin et al. 2011), it remains unclear how repeated administration over several weeks would affect disuse-related motor system changes.
As mentioned earlier, chronic SCI patients (especially motor-complete also called ASIA-A or ASIA-B patients) experience often life-threatening health problems also referred to as ‘secondary complications’ including motor system changes reported here also in this paraplegic mouse model. Using combination therapy, we obtained preliminary data suggesting that repeatedly-treated paraplegic mice can partially prevent some pathophysiological motor system changes found after SCI (Guertin et al., 2011).
Video images of a paraplegic mouse placed on a treadmill 15 minutes following administration of a CPG-activation tritherapy. Involuntary movements were generated for approximately 30 to 45 min. Then a complete return to complete paraplegia occurred.
Subcutaneous administration (several times per week) of a first-generation combination treatment was found, upon each injection (within 15 min), to repeatedly induce temporarily (during approx. 30-45 min) episodes of weight bearing stepping in non-assisted paraplegic mice at least during one month.
Regarding body weight values, combination therapy-treated paraplegic animals progressively displayed a moderate increase in weight suggesting that repeated administration of this combination therapy was well-tolerated (i.e., a loss of weight would have suggested toxic effects and additional health problems). No significant difference was found in bone mineral density (BMD) values in femoral bones of tritherapy-treated vs. placebo-treated paraplegic mice. Post-mortem examination of muscle size (whole surface area and fiber cross-sectional area or CSA) measured from cryostat transverse sections prepared from two hindlimb muscles, soleus (SOL) and extensor digitorum longus (EDL), was performed to assess the effect of combination therapy-induced training on muscular atrophy normally found after SCI. We found values corresponding with larger muscles and muscle fibers in the combination therapy-treated compared with the placebo-treated paraplegic animals. Sol values increased by 24% in combination therapy-treated paraplegic mice (0.61 ± 0.05 mm2) compared with placebo-treated ones (0.49 ± 0.03 mm2, fig. 3A). Comparable results were found in EDL (combination therapy-treated 0.91 ± 0.03 mm2 vs. placebo-treated 0.77 ± 0.06 mm2)(not shown). At the cellular level, comparisons between combination therapy-treated and placebo-treated animals revealed that type I fiber CSA values non-significantly changed whereas type II fiber and intermediate fiber (type I + II labeled) CSA values significantly increased subsequently both by 8% (Fig. 3C,\n\t\t\t\t\t3D). An analysis of muscle fiber-type ratios (i.e., proportion among all fibers of type I, type II or type I + II fibers) indicated that no significant changes were found between groups. Subpopulations of red blood cell (RBC) constituents were assessed and compared between groups. Levels of RBC, platelet, hemoglobin and hematocrit were significantly increased by 11%, 19%, 10% and 10%, respectively, in combination therapy-treated vs. placebo-treated paraplegic animals.
All in all, these results revealed that pharmacological activation of the CPG four times per week during 1 month can prevent anemia and prevent partially muscle atrophy. Circulatory systems were not further examined in this study. On the other hand, this study showed that bone loss typically occurring post-transection in this animals can not be prevented in these conditions. Altogether, it is suggested that training conditions or treatments may have to be optimized for further physiological effects on all parts of the motor system.
Along this idea, we recently conducted a study where paraplegic animals received an anabolic agent, namely clenbuterol, in addition to tritherapy-induced locomotor training. We found that tritherapy-treated paraplegic mice with or without clenbuterol treatment displayed significant locomotor function recovery during 2 months upon each administration of the CPG-activating therapy (Fig.7). To further characterize movements induced by the tritherapy-training, angular excursion at the hip, knee and ankle, as well as movement amplitude values were analysed. Typical examples of hindlimb kinematics are shown in figure 7. Hip, knee and ankle angular displacement showed similar patterns in intact, tritherapy-trained alone and tritherapy-trained + clenbuterol paraplegic animals. Untrained paraplegic animals displayed a consistent lack of angular excursion at the hip level although some displacements were found at the knee and ankle levels (hip: 85°, knee: 30-47°, ankle: 28-125°). Hindlimb movement amplitude values measured by calculating toe displacement in X and Y axis (step “length” and “height”) revealed that intact mice had greater step length values than both tritherapy-trained paraplegic groups. On the other hand, both groups of tritherapy-trained paraplegic animals showed similar step length values which were significantly greater than those in untrained paraplegic mice. The coefficient of variation (CV) was higher in untrained mice. Intact, tritherapy-trained and tritherapy-trained + clenbuterol paraplegic mice showed similar step height values. However, differences were found in the variability of the step height, as shown by CV, where intact animals displayed less variability than the other groups of tritherapy-trained animals. No Y axis movement amplitude was observed in paraplegic untrained mice since no weight-bearing movement are normally expressed spontaneously. Overall, a significant increase in performances over time was observed in tritherapy-trained groups of paraplegic mice movement kinematic values were comparable with those from intact animals.
Kinematic analyses in intact (non-Tx), paraplegic (Tx) untrained, paraplegic tritherapy-trained and tritherapy-trained and treated with clenbuterol. Step parameters from both tritherapy-trained paraplegic mice were similar with those from intact animals suggesting that the tritherapy appropriately restored episodes of locomotor movements.
Femoral BMD and BMC values were measured in order to address whether tritherapy-training alone or combined with clenbuterol can prevent or at least reduce bone loss normally found in untrained paraplegic mice. However, in all groups of paraplegic animals, important losses were found. Untrained paraplegic mice (BMD: 0.0767 ± 0.0010 g/cm2, BMC: 0.0381 ± 0.0008 g) and tritherapy-trained paraplegic animals (BMD: 0.0766 ± 0.0011 g/cm2, BMC: 0.0378 ± 0.0009 g) showed comparable values whereas in paraplegic trained + clenbuterol groups, femoral BMD (0.0731 ± 0.0012) and BMC (0.0349 ± 0.0009) further decreased.
Morphometric analyses of soleus and EDL were performed in order to further characterize specific muscular property changes in all groups. Muscle CSA, fiber type-specific CSA and relative distribution values were analysed. For soleus CSA, untrained and tritherapy-trained paraplegic mice had significantly lower muscle CSA values than intact animals and tritherapy-trained + clenbuterol paraplegic groups. However soleus CSA in untrained paraplegic mice was not significantly lower than tritherapy-trained paraplegic animals. For EDL, in contrast with muscle mass changes, CSA values showed statistical differences between groups. Tritherapy-trained + clenbuterol paraplegic mice showed higher EDL CSA values than all the other groups. Untrained and tritherapy-trained paraplegic mice had lower CSA values than intact animals.
Femoral BMD and BMC in intact (non-Tx), paraplegic (Tx) untrained, paraplegic tritherapy-trained and tritherapy-trained and treated with clenbuterol. Unfortunately, bone loss was not prevented in both tritherapy-trained paraplegic mice compared with intact animals suggesting that the tritherapy with or without clenbuterol failed to restore bone properties.
Soleus and EDL cross-sectional area values in intact (non-Tx), paraplegic (Tx) untrained, paraplegic tritherapy-trained and tritherapy-trained and treated with clenbuterol. Although encouraging anti-atrophying effects were found in tritherapy-treated paraplegic mice, only paraplegic animals that received both clenbuterol + tritherapy displayed a complete restoration of muscle size (even a relative hypertrophic effect was induced compared with intact animals).
More differences were found when analysing individually fiber type-specific CSA values. Specifically, for soleus fiber types, all three fiber types from tritherapy-trained + clenbuterol paraplegic animals displayed larger CSA values than all other groups (type I: 1656.7 ± 80.8 µm2, type II: 987.2 ± 16.7 µm2, hybrid: 1145.5 ± 18.0 µm2). Conversely, untrained paraplegic mice displayed the lowest soleus fiber type CSA of all groups (type I: 783.1 ± 15.1 µm2, type II: 753.2 ± 9.1 µm2, hybrid: 750.0 ± 8.1 µm2). In EDL, type II fiber CSA differences between groups were similar to soleus type II (intact: 1063.5 ± 15.9 µm2, paraplegic untrained: 908.1 ± 11.4 µm2, paraplegic trained: 963.4 ± 10.9 µm2, paraplegic trained + clenbuterol: 11.65.2 ± 17.9 µm2).
These findings provided proof-of-concept data strongly supporting the idea that physical activity can prevent or restore motor system adaptations normally expressed after SCI. However, that study was exploratory and thus, it remains unclear the extent to which physical activity elicited with this pharmacological approach can extensively prevent or reverse secondary complications. Although anemia and partial muscle atrophy were prevented in CPG-activating tritherapy-trained paraplegic mice, addition of anabolic aids such as clenbuterol appeared to synergistically affect positively the motor system in paraplegic mice (complete reversal of atrophy, complete lack of anemia, etc.). Effects on other elements of the motor systems such as blood vessels (e.g., deep vein size) or skeleton remain to be explored or improved. From a scientific perspective, it remains also to be determined clearly what role physical inactivity may play on motor system adaptations post-SCI and corresponding health problems in humans. This said, motor system changes post-SCI obtained in this murine model was found to resemble those typically encountered in patients with SCI or disuse. It may therefore be useful to further study basic cellular mechanisms underlying these changes of the musculoskeletal systems in these conditions. It may also serve to accelerate the development of new therapeutic strategies aimed at reducing or preventing completely all musculoskeletal and biomechanical changes in SCI patients or in patients suffering of disuse or immobilization.
Microorganisms are among the most important sources of poor indoor air quality, and contamination of indoor air by microbial pollutants is being increasingly recognized as a public health problem and as one of the factors contributing to sick building syndrome. Bioaerosols, such as those comprising fungi, bacteria, and viruses, in indoor air can cause allergic and infectious diseases, respiratory problems, and hypersensitivity reactions. People who are sensitive to indoor environmental problems complain of a wide variety of symptoms, ranging from headache, tiredness, nausea, and sinus congestion to eye, nose, and throat irritations [1].
\nAlthough invisible to the naked eye, the atmosphere is populated by a diversity of microorganisms, including bacteria, fungi, algae, and protozoa. Researchers have estimated that the total bacterial count within the troposphere layer ranges from 1 × 103 to 1 × 105 cells/m3. Dust is formed during the passage of organic and inorganic particles from external and internal resources, which subsequently aggregate and precipitate. House dust, for example, consists of cotton fibers, hair, bacteria, molds, and remaining paint particles [2, 3]. The findings of a previous study have indicated that the average number of fungi contaminating 820 indoor air-conditioning units was 1252 CFU/m3, with range of 17–9100 CFU/m3. In addition, Baxter [4] found that the average number of spores isolated from 85 buildings was 913 cells/m3, ranging from 68 to 2307 cells/m3. Daily and seasonal numbers of contaminant microorganisms in the air vary and depend primarily on environmental factors, such as vegetation, human activities, and seasonal fluctuations [5]. Most of these microorganisms are bacteria and fungi [6].
\nThese microbial contaminants affect the residents of enclosed and humid buildings, particularly in the case of toxic hygrophytic fungi, such as Phoma sp., Exophiala sp., Aureobasidium pullulans, Acremonium sp., and Sporobolomyces, that are frequently isolated from the cooling pipes of air-conditioning systems. Gram-negative bacteria and their toxins are also isolated from leaks in air-conditioning pipes. Yang [7], for example, identified Legionella pneumophila, which is the causal agent of legionnaires disease, as a dominant bacterium in the water leaking from cooling systems. In addition, Pseudomonas aeruginosa, which has also been isolated from water leaking from air-conditioning systems, is an opportunistic bacterium responsible for several diseases. Many studies have proven that the heating, ventilation, and air-conditioning (HVAC) systems can become contaminated with organic pollutants, bacteria, and fungi, as well as by particulate matter derived from mice, insects, and nematodes. The bacteria and fungi colonizing these systems tend to saprophytic and thrive in areas that meet their environmental requirements [7]. Fungi have been proven to be a source of airborne contamination in air-conditioning systems [8], including Alternaria, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus ochraceus, Aspergillus versicolor, Botrytis cinerea, Cladosporium herbarum, Epiccocum purpurascens-sterilia, and Penicillium spp., among which A. fumigatus, which has been isolated from air-conditioning filters, is responsible for many dangerous infections. With regard to bacteria, Propionibacterineae, Staphylococcus, Streptococcus, and Corynebacterineae (17, 17.5, 20, and 3%, respectively) have been detected in aeration pipes and air filters installed in indoor areas [9, 10]. In addition small percentages of species from the genera Fusobacterium and Veillonella (0.02 and 0.1%, respectively), which are associated with the mouth cavity and saliva, have also been identified as air-conditioning system contaminants [11, 12, 13].
\nWith a view toward providing clean indoor air, several studies have been conducted to investigate measures that can be used to control the levels of microorganisms that colonize filtering, heating, ventilation, and air-conditioning systems. In this regard several types of air filters have been studied with the aim of preventing the penetration of particles. However, although high-efficiency particulate air (HEPA) filters are widely used in hospitals, Aspergillus-associated infections continue to occur [14]. Currently, most indoor air-conditioning systems contain internal filters that extract microorganisms from the air (Figure 1). However, these microbes often remain viable and can be returned to the surrounding atmosphere under certain circumstances, such as inefficient operation, during periods of maintenance, or due to temporary malfunction [15].
\nAccumulated dust on discarded polyester filters.
It is widely acknowledged that air-conditioning filters do not remove all the particles from the air. Even the use of HEPA filters will not completely eliminate the problem of microbial contamination, as this material will only retain particles of a minimum of 3 microns in size. Thus, dust particles with sizes smaller than 3 microns will pass through unhindered. Furthermore, when the filters become excessively wet, they can provide a fertile environment for the proliferation of molds and bacteria [16, 17].
\nIn this chapter, several new topics related to environmentally sustainable buildings were presented, as it clearly describes the potential impact of HVAC systems on the indoor air quality with the aim to enhance the healthy buildings. The chapter is structured as follows: besides the Introduction (Section 1), Section 2 introduces the principle of air filtration, Section 3 is concerned with presenting the traditional air filters, Section 4.5 demonstrates a comparison between the most common and modern HVAC filters, Section 6 provides the impact of HVAC filters on indoor air quality, and Section 7 is concerned with presenting several results for research progress about the relationship between microbes and traditional filters and microbial colonization of the types of filters commonly used in air-conditioning systems.
\nThere are five different collection mechanisms that determine air filtering performance: straining, interception, diffusion, inertial separation, and electrostatic attraction.
\nThe first of these mechanisms applies mainly to mechanical filters and is influenced by particle size. Figures 6 to 10 illustrate the five mechanical principles of particle capture and their contribution to the retention of particles of different sizes.
\nStraining (sieving) occurs when the opening between the media components (e.g., fibers, screen mesh, and corrugated metal) is smaller than the diameter of the particle the filter is designed to capture. This principle spans across most filter designs and is entirely related to the size of the particle, media spacing, and media density (Figure 2).
\nModel of straining (sieving) mechanism, depends on the space between the fibers.
Interception occurs when a large particle, because of its size, collides with a fiber in the filter that an air stream is passing through (Figure 3).
\nModel of interception effect mechanism, depends on the collision between the fiber and the particle passing through the filter.
Diffusion occurs when the random (Brownian) motion of a particle causes that particle to come into contact with a fiber. When a particle vacates an area within the media, by attraction and capture, it creates an area of lower concentration within the medium into which another particle diffuses, only in turn to be captured itself. To enhance the likelihood of this attraction, filters employing this principle operate at low media velocities and/or high concentrations of microfine fibers, glass, or otherwise (Figure 4).
\nModel of diffusion mechanism, depends on the motion of the particle causing contact with a fiber.
Inertial separation is based on a rapid change in air direction and the principles of inertia to separate particulate matter from the air stream. Particles moving at a certain velocity tend to remain at that velocity and travel in a continuous direction. This principle is normally applied when there is a high concentration of coarse particulate matter and in many cases represents a pre-filtration stage prior to the passage of air through higher-efficiency final filters (Figure 5).
\nModel of inertial separation mechanism, depends on the collision between the fiber and the small particles for reducing its velocity.
Electrostatic attraction is obtained by charging the media as a part of the manufacturing process (Figure 6). However, it plays a minor role in mechanical filtration. After fiber contact is made, smaller particles are retained on the fibers by a weak electrostatic force. The force may be created through a manufacturing process or be dependent upon airflow across media fibers. The force is eradicated as media fibers collect contaminants that act as an insulator to a charge. Electrostatic filters, which are composed of polarized fibers, may lose their collection efficiency over time or when exposed to certain chemicals, aerosols, or high relative humidity. A decrease in pressure in an electrostatic filter generally increases at a slower rate than it does in a mechanical filter of similar efficiency.
\nModel of electrostatic attraction mechanism, depends on charging the fiber to retain the small particles by a weak electrostatic force.
Inertial separation and interception are the dominant collection mechanisms for particles greater than 0.2 μm in size, whereas diffusion is dominant for particles less than 0.2 μm in size.
\nAs mechanical filters become loaded with particles over time, their collection efficiency and reduction in pressure typically increase. Eventually, the decrease in pressure significantly inhibits airflow, and when this occurs, the filters must be replaced. For this reason, a decrease in pressure across mechanical filters is often monitored, as this can provide an indication of when the filters need to be replaced. Thus, unlike mechanical filters, a decrease in the pressure of electrostatic filters is a poor indicator of the need to change filters. When selecting an HVAC filter, these differences between mechanical and electrostatic filters should be borne in mind because they will have an impact on filter performance (collection efficiency over time), as well as on maintenance requirements (changeout schedules).
\nHumans consume approximately 30 liters of oxygen per hour. Hence, our requirement for air is relatively small: 0.15 m3/h. However, because we also produce carbon dioxide, our bodies require approximately 5 m3/h of fresh air in order to maintain carbon dioxide concentrations below life-threatening levels. When installing an air-conditioning system, it is advisable to determine the amount of air needed, and this will generally be set at between 15 and 20 m3 per individual per hour. However, larger volumes of air might be necessary for managing temperature or drawing off polluted air.
\nEnsuring that air is free of dust and aerosols is not only important for maintaining buildings and their interior but also essential for maintaining the health and well-being of the human inhabitants.
\nThis may be due to the higher foot traffic during business hours. The air output of these places is relatively high, and the cleaning of air-conditioning units may prove difficult, which could favor microbial growth and increased accumulation of dust on filters and in ducts. With respect to building contamination, it has been found that hospitals tend to have higher levels of contamination than other types of building examined. Given that hospitals are permanently inhabited by patients, this accordingly increases the potential for contamination and possibly infection by opportunistic pathogens [18, 19].
\nAl-Abdalall and Al-Abkari [20] examined the most commonly used filters incorporated in air-conditioning systems, namely, sponge, polyester, and HEPA, in order to assess the efficiency with which these filters can prevent the passage of fungi and bacteria. They accordingly found that complex filters were the most efficient in terms of purifying air, with efficiency rates up to 91.8% for bacteria and 100% for fungi. Sponge filters were deemed to be the least efficient filters, with estimated filtration rates of 2 and 50% for bacteria and fungi, respectively. This difference can probably be explained in terms of the passage of air through filters, with filters containing smaller pores being able to trap the larger cells of bacteria or fungi more efficiently. In other words, sponge filters are less efficient for air purification due to the large filter pores, whereas the filters of HVAC systems are able to capture particles smaller than 0.5 microns and prevent all particles with sizes greater than 3 microns from passing through [21].
\nIn this regard, there are a number misconceptions concerning the relationship between filter efficiency and particle size, and in order to resolve this issue, a number of companies have developed certain filter-related standards based on particle counts at the most penetrating particle size (MPPS). The European Standard applies to HEPA ULPA filters used in the field of ventilation and for technical processes (e.g., for clean room technology or applications in the nuclear and pharmaceutical industries).
\nMany indoor air quality problems can be solved or avoided by cleaning or replacing air filters on a regular basis. Since using air filters is one of the most common methods of purifying air, it is recommended that filters be checked at 3-month intervals or that arrangements are made for a certified technician to change the filter at the beginning of each season.
\nFilters tend to become clogged, and once their holding capacity has been reached, particulate matter tends to be released downstream in the system and into the heat exchanger and can thereby cover the interior of the ductwork and the blower motor. This matter can subsequently cause problems and malfunctions in the mechanical and electrical parts of the system, resulting in high repair costs and even the need for replacement. The dispersed matter will circulate back into the house, potentially resulting in the proliferation of molds and other fungi. Dirty filters can also have a detrimental effect on energy consumptions due to impeded airflow, resulting in an increased in fan runtime.
\nAccording to the American Society of Heating, Refrigerating, and Air-Conditioning Engineers (ASHRAE) Standard 52.2-2007 [22], the performance of an air filter is determined by measuring the particle counts on both the upstream and the downstream sides of the air filter device being tested. Through provided capture efficiency values for a range of particle sizes, it facilitates the selection of a filter that has the best efficiency with regard to removal of the target contaminant.
\nTo simplify filter selection, the Standard defines a minimum efficiency reporting value. The MERV is a single number that simplifies the filter selection process by providing the specifier, or the user, with a single value of specification for filter selection. For most filters with mechanical-based filter operation, this number will most probably be a minimum value at installation and throughout the life of the filter. The particle size ranges specified by Standard 52.2 and an illustration of how to read an ASHRAE 52.2-2007 [22] test report are shown in Tables 1 and 2 and Figure 5, respectively (Figure 7).
\nRange | \nSize (microns) | \n
---|---|
1 | \n0.3–0.4 | \n
2 | \n0.4–0.55 | \n
3 | \n0.55–0.7 | \n
4 | \n0.7–1.00 | \n
5 | \n1.00–1.30 | \n
6 | \n1.30–1.60 | \n
7 | \n1.60–2.20 | \n
8 | \n2.20–3.00 | \n
9 | \n3.00–4.00 | \n
10 | \n4.00–5.00 | \n
11 | \n5.00–7.00 | \n
12 | \n7.00–10.00 | \n
Particle size ranges of Standard 52.2.
Example particle | \nSize (μm) | \n
---|---|
Hair | \n20–200 | \n
Pollen | \n10–100 | \n
Spores | \n10–25 | \n
Toner | \n5–20 | \n
Oil fog | \n0.3–5 | \n
Bacteria | \n0.2–25 | \n
Tobacco smoke | \n0.01–1 | \n
Virus | \n0.002–0.05 | \n
Particle size ranges of common pollutants specified by Standard 52.2.
How to read an ASHRAE 52.2-2007 test report.
Unfortunately, filters that use the principle of electrostatic attraction can “fool” the test by providing a high MERV during tests. However, due to the loss of electrostatic attraction during operation, a much lower value is obtained during application. Hence, the user may not be getting the particle removal efficiency that they originally specified.
\nMultiple studies have shown that coarse-fiber media (charged synthetic media), unlike fine-fiber media (fiberglass media), perform differently in real-life applications. Coarse-fiber media depends on an electrostatic charge to achieve the published filter efficiency. When atmospheric air, in which 99% of the particulate matter less than 1.0 micron in size, passes through a filter, the very fine particulate matter will dissipate the charge, and the filter rapidly loses efficiency.
\nQian [23] isolated Streptophyta from dust samples collected from the filters of air-conditioning systems at a rate of 45%, whereas the rate in the indoor air was found to be only 2.4%, which provides an indication of the efficiency of HVAC filter systems (preventing particles sizes that are larger than 3 μm).
\nThere are six types of filters, which are briefly described below.
\nThe main advantage of fiberglass filters is they are very cheap, easy to install, and readily available in stores. Accordingly, although they have a lifespan of only 1 month, replacing them on a monthly basis would not pose an inordinate financial burden. Unfortunately, they are not particularly effective in terms of trapping particles.
\nThese filters are more effective with regard to trapping dust than fiberglass filters. They can trap approximately 45% of airborne debris. Their MERV rating typically lies somewhere between 10 and 13. They also have a 1-month lifespan but tend to be more expensive than fiberglass filters.
\nThese filters use electricity to attract charged particles, which are trapped internally. They are very efficient at trapping dust particles and debris and have a 6-month lifespan.
\nThey are the most economical type of filter, which can be removed and cleaned as directed, dried, and then reinstalled. Furthermore, they do not need replacing at monthly intervals. These filters can prevent the passage of debris and tend to function better when dirty. However, they have a MERV rating of only 1–4.
\nThese filters comprise a cardboard frame and filter material. As their name implies, it is necessary to replace them when they become dirty.
\nHEPA filters are considered the best type of filter because they trap even the smallest particles and keep premises smelling fresh. They can capture up to 97% of all particulate matter and remove all allergens from indoor air.
\nThe type of air filter is the first factor people take into consideration before deciding on which air purifier to purchase.
\nAir filters and electrostatic filter cleaners are typically rated according to the minimum efficiency reporting value, commonly known as the MERV rating. The MERV scale is a measurement scale developed in 1987 by ASHRAE to rate the effectiveness of air filters, which determines efficiency in terms of the size of particle that the filter will capture. Values vary from 1 to 16, with a higher number indicating the greater efficiency of the filter in trapping airborne particles.
\nRecently, UV lights have been widely employed in the ducts of HVAC filtration systems. These lights facilitate effective and inexpensive control and solve the problem of microbial outgrowth in HVAC systems, eliminating up to 99.9% of the microorganisms and destroying airborne viruses, bacteria, and fungi. The types and quantities of microorganisms killed depend on the length of exposure and the output of the lamps. Nowadays, more advanced UV lights, such as air scrubbers, are employed, which can kill both airborne viruses and bacteria and those growing on surfaces.
\nThere are two main types of UV lights used for HVAC systems, the most common of which are coil sterilization UV lights, which are installed near the return ducts, so as to kill mold that may grow on the air handler coil. These UV lights operate 24 h a day and eliminate the need for removing mold from the air handler coils. The second main type of UV lights is air sterilizer UV lights that function by sterilizing the air passing through the return ducts [24].
\nAlso referred to as charcoal-impregnated air filters, these types of filters are used to effectively remove odors and fumes from the air during the air recirculating process.
\nCommercial activated carbon filters provide high-efficiency odor, fume, and gas removal and are fabricated using the finest quality coatings, including bulk air filter media and pads cut to size, pleats, panels, and high-density granular carbon packs.
\nSynthetic media substrates, such as non-woven polyester, are impregnated with finely ground coatings, including activated carbon, zeolite, or alumina, and a heat set to retain these coatings even when the activated carbon filter media is rinsed or vacuumed. Just as a sponge soaks up water, the media of activated carbon air filters absorb odors and fumes. Moreover, the odor-causing molecules are permanently removed from the air, rather than simply being masked with a different odor.
\nThe rate of adsorption depends on the relationship between the pore structure, or surface area, and the shape of the contaminating molecules. Activated carbon filters are disposable air filters, and once they have become saturated with odors, fumes, or gases, after approximately 3 to 6 months of use, they must be replaced. The amount of activated carbon required will depend on the amounts of odors, fumes, or gases to be removed [24].
\nDeodorizing air filters use acidified titanium, activated carbon, ceramic fiber, pulp, and other advanced materials that are prepared using a variety of rigorous refinement processes. They function by purifying the air and maintaining air fresh and show superior efficacy when used in conjunction with UV irradiation [24].
\nAntibacterial filters are prepared by incorporating a bactericidal substance in the filter media. However, doubts remain regarding the effectiveness of these filters. One type is prepared by simply spraying the additive onto the surface of the filter medium, and therefore effective coverage is often not achieved, and not all of the filter layers will kill bacteria. A second type is prepared by application of a bacteriostatic agent, which does not kill the bacteria and may indeed promote the development of drug resistance among the bacteria. A third type may generate certain gaseous substances or odors that are potentially harmful to humans. Furthermore, it should be emphasized that it is difficult to capture bacteria on the windward side of the HEPA filters fabricated from inorganic materials, into which the bacteria may even penetrate. However, these microorganisms are only likely to survive under suitable conditions of temperature and humidity. Accordingly, the efficacy of these antibacterial filters remains inconclusive, and ASHRAE has recommended that HVAC systems incorporating antibacterial filters should be used with caution, so as not to produce any additional chemical pollution within indoor environments [24].
\nAlthough air filters often show excellent removal efficiency with regard to pathogens, the captured microorganism can remain viable within the filter and subsequently grow and become re-dispersed in the air flow, thereby generating a secondary source of pollutants and unpleasant odors. In an effort to resolve this issue, a large number of studies have been conducted with the objective of improving the effectiveness of air filters with antibacterial properties, and some of these studies have demonstrated that such air filters can be successfully prepared by incorporating inorganic nanoparticles and natural plant extracts [25].
\nKim [26] also evaluated the efficacies of various functional filters coated with antimicrobial chemicals in deactivating representative microorganisms on filters or as bioaerosols. Specifically, they examined the effectiveness of functional filters coated with different chemicals, including ginkgo and sumac; Ag-apatite and guanidine phosphate; SiO2, ZnO, and Al2O3; and zeolite, using a model ventilation system to evaluate the efficiency in which bacteria (Escherichia coli and Legionella pneumophila), bacterial spores (Bacillus subtilis spore), and viruses (MS2 bacteriophage) were removed. Their result showed that although the functional filters could facilitate the biological removal of various bioaerosols, physical removal was minimal. Appropriate use of chemical-coated filter materials could reduce exposure to these agents.
\nElectrostatic filters: Static electricity attracts dirt and dust to vertical and overhead surfaces. The static is often generated when two surfaces rub together and are then separated. Electrostatic filters generate a static electrical charge on all particles in the air that passes through eight filter layers. The discharged particles are then attracted to collector plates with an opposite electrical charge. These filters have the advantage of being washable.
\nHigh-efficiency particulate air filters: HEPA filters have a strong particle-trapping capacity that facilitates the removal of a high percentage (99.97%) of airborne particles that pass through an air purifier and accordingly meet US government standards. This contrasts with the 60–90% efficiency of medium filters [27]. Furthermore, HEPA filters perform significantly better than electrostatic air cleaners, in which filtering is based on ionic processes. HEPA filters are therefore often used in medical facilities and in households in which the residents suffer from severe allergies.
\nPhotocatalysts are nanoscale metal oxide materials (commonly titanium dioxide) that are applied to substrate surfaces, forming a film after drying under the action of light. They have a strong catalytic degradation function and can be used to degrade hazardous atmospheric gases. They can also be used to effectively kill a variety of bacteria, with an antibacterial rate of 99.99%. Furthermore, toxins released during the degradation of bacteria and fungi can be rendered harmless. These catalysts also have a range of other properties, including deodorant and dirt removal functions.
\nExposure to bioaerosols can cause various adverse health effects, including infectious and respiratory diseases and hypersensitivity. Consequently, controlling the exposure to bioaerosols constitutes an important aspect of disease control and prevention.
\nPhotocatalytic oxidation systems use a UV light source and a titanium dioxide photocatalyst to produce oxidants that destroy gaseous contaminants. When the photocatalyst is irradiated with UV light at wavelength of 254–365 nm, a photon from the light excites a catalyst electron in the valence band to jump to the conduction band, leaving a hole. This photocatalytic oxidation process converts organic pollutants into carbon dioxide and water. Using this technique, pollutants, particularly volatile organic carbons, are preferentially adsorbed on a catalyst surface and oxidized. The hole generated by photocatalysis can further react with surrounding water to produce a hydroxyl radical (ÆOH), whereas the electron in the conduction band reacts with oxygen to yield a superoxide radical anion (ÆO2). These radicals can attack the cell membranes of microorganism, thereby causing the release of K+ ions, RNA, proteins, and other important components and eventually resulting in cell death [28]. Given these properties, researchers have applied photocatalytic oxidation to many substrates and achieved impressive results, indicated by the affective control of test microbes.
\nTo date, however, photocatalytic oxidation has yet to be applied to HEPA filters in HVAC systems [27]. HEPA filters have been mandated for use in the removal of airborne microorganisms in many codes adopted in the field of healthcare, including the American Institute of Architects Guidelines for Design and Construction of Hospital and Health Care Facilities (AIA Guidelines), the American Society of Heating, Refrigerating and Air-Conditioning Engineers standards, the Joint Commission on Accreditation of Healthcare Organizations Environment of Care standards, the Centers for Disease Control and Prevention (CDC) guidelines, and recommended practices [29]. Although HEPA filters can efficiently capture aerosolized microorganisms, the area downstream of the filter can become a breeding ground for microbes. Under conditions of suitable temperature and humidity, microbes retained within a filter can multiply using particulates adhered to the filter as a food source, and the microbial progeny can ultimately disperse into the filtered air [30, 31]. Thus, instead of being an apparatus to control air quality, these systems can potentially become a source of pathogens. Efforts have accordingly been made to eliminate the breeding ground problem. For example, Goswami [32] examined four microbial species (Aspergillus niger, Penicillium citrinum, Staphylococcus epidermidis, and Bacillus subtilis) that are representative of the genera most commonly detected in hospitals in Thailand, a country characterized by a hot and humid climate, with an average temperature of 27°C and average relative humidity ranging from 62 to 84% [33, 34].
\nVery high relative humidity not only reduces the probability of microorganisms coming into contact with hydroxyl radicals but also provides sites conducive to microbial survival. Excessive amounts of water can also occlude the reactive sites of filter surfaces and subsequently reduce the photocatalytic oxidation efficiency [32, 34, 35]. Hence, the effect of relative humidity has been investigated.
\nChuaybamroong [27] examined the application of photocatalytic oxidation to HEPA filters for disinfection of airborne microorganisms. Experiments were conducted at two TiO2 loadings on HEPA filters irradiated with UV-A under two relative humidities. They assessed the inactivation of two fungal (Aspergillus niger and Penicillium citrinum) and two bacterial (Staphylococcus epidermidis and Bacillus subtilis) isolates and found that, on average, 60–80% of microorganisms retained on a photocatalytic filter were inactivated, although in the case of S. epidermidis, 100% inactivation was observed.
\nThese authors suggested that high humidity may induce the reactivation of organisms, whereas water may occupy most of the TiO2 sites, leaving fewer available sites for microbial colonization. These conjectures are consistent with the opinions of [36], who noted that although the presence of water vapor enhances the likelihood of hydroxyl radical formation, at certain humidity levels, radical formation would not increase with increasing water vapor and even decrease due to the occlusion of adsorption site on the TiO2 surface. Consequently, high humidity would decrease filter efficiency. Furthermore, Peccia [37] indicated that high levels of relative humidity may promote changes in the biopolymers within microbial cells, including cell wall components, or alter protein structure, thereby affecting DNA repair enzymes, and hence could protect the microorganism from desiccation and/or attenuate the incident UV irradiation.
\nFiltration technology is currently an integral aspect of air purification techniques that focus on particulate matter, the most common examples of which are fibrous filters [38]. The use of glass fiber filters is another mature filtration technique with a proven high efficiency (99.0%), similar to that of the HEPA filters [39]. Furthermore, wire mesh filters can also provide good filtration efficiency down to sizes of 2–10 μm [40].
\nOn the basis of particle filtration efficiency, air filters can be divided into four types: prefilters, medium filters, HEPA filters, and ultralow particulate air (ULPA) filters [41]. The filtration efficiency of ULPA filters is greater than 99.999%, with particles of diameters down to 0.12–0.17 μm being effectively trapped [42]. Similarly, HEPA material has a strong ability for trapping particles and can remove 99.97% of particulate matter, smog, and bacteria with sizes down to 0.3 μm (Figure 8). In contrast, the efficiency of medium filters is only between 60 and 90% [27, 43]. Most air purifiers currently on the market incorporate HEPA filters, as these are internationally recognized as the most efficient filters, capturing particles of different diameters. HEPA filters are designed to be over 99.99% efficient and are used in a diverse range of situations, including in theaters, hospitals, respirators, and vehicles [44]. Furthermore, filtration based on the use of non-woven nano-fiber material is an emerging filtration technique with an extremely high efficiency that is comparable to, or even superior to, HEPA-based filtration in the smaller particle size range [45].
\nA standard filter traps large dust particles in internal structure of an air purifier. HEPA filter captures microbes.
According to the Environmental Protection Agency (EPA), pollutant levels may be two to five times higher indoors than outdoors, which indicates that the poor quality of indoor air is mainly attributable to the inefficient circulation of air. In regions characterized by hot summer, there is a heavy reliance on air-conditioning systems for maintaining comfortable conditions during the hot summer months. Accordingly, windows tend to remain shut, and little fresh air enters into our homes and places of work. The EPA warns that high temperatures and humidity can increase the concentrations of certain pollutants, with young children and the elderly being at particular risk from the detrimental effects of indoor air pollution.
\nMechanisms for trapping dust in air using a standard filter, killing almost of all airborne microbes using UV lamps, and removing fine particles (dust) and died microbes using a high-efficiency particulate air filter (Figure 8).
\nGiven that dirty HVAC units have been proven to be less efficient, it is essential that air-conditioning ducts are periodically cleaned through employing an air duct cleaning service. Such cleaning should include complete care of the internal elements of the HVAC unit.
\nDuctwork would only be essential if there has been renovation, asbestos abatement, lead paint removal, or a significant accumulation of dust debris. Cleaning would be considered essential in the presence of the following: animal feces, mold, foul odors, noticeable debris, or pet hairs. Furthermore, if occupants suffer from an unexplained allergy, then it would be advisable to consider cleaning. Abe [46] noted that it is possible to remove fungi and bacteria from filters by washing with water and detergent; however, if the fan and heat exchanger are also contaminated, specialist cleaning would be required (Figure 9).
\nDucts should be cleaned periodically.
Kujundzic [47] mentioned that cleaning room air could contribute to reducing the levels of particulate matter within the home and that this can be achieved by using filters that retain the filtered particles.
\nMold is a pervasive problem, of which many property owners are fully unaware. In the Eastern Province of Saudi Arabia, the average annual humidity level is approximately 74%, but can be notably higher during certain times of the day and year. Many types of mold require a humidity of only 50% to commence growth, and air-conditioning systems are a common source of mold in many households. One of the causal factors in this respect is the fact that HVAC systems do not operate continuously, which can result in a fluctuation in humidity levels. The EPA warns that if an HVAC system is turned off before occupants perform tasks such as mopping, the humidity levels can suddenly surge and cause moisture and mold problems. If an HVAC system is improperly programed (which is a common problem), then the air-conditioning system may cycle off when the air is cooled but before it has had time to dry sufficiently, causing moisture problems. HVAC maintenance issues can be a further source of mold-related problems, such as when excessive moisture accumulates on the air-conditioning coils, resulting in the growth of mold. This mold can subsequently be blown through the air-conditioning ducts and released into the surrounding air. Holes or gaps in air ducts can also lead to the formation of condensation, which creates a perfect breeding ground for mold to grow. Such mold can cause numerous health-related problems, including respiratory problems, skin rashes, and allergic reactions [17].
\nIn a study designed to examine the efficiency of various filters used for trapping microorganisms, Al-Abdalall and Al-Abkari [20] isolated the bacteria and fungi colonizing air-conditioning systems in different types of buildings during each of the four season s, in the provinces of Dammam and Qatif in eastern Saudi Arabia, and determined the respective frequency distributions. The air-conditioning systems were found to be contaminated by different types of bacterial and fungal species. Specifically, the isolated bacteria included Serratia liquefaciens, Bacillus pumilus, Bacillus cereus, Bacillus subtilis, Staphylococcus lentus, and Oligella ureolytica, whereas the common fungal taxa included Cryptococcus laurentii, Aspergillus niger, Aspergillus flavus, Cladosporium sp., and Rhizoctonia sp. Ironically, the findings of the study indicated that buildings that were in good condition were those likely to have the highest levels of microbial contamination (Figure 10).
\nMicroorganisms collected by swabbing an air-conditioning duct and using the swabs to inoculate a fungal growth medium. The left-hand panel shows Aspergillus fumigatus growth on inoculated medium. The right-hand panel shows the mycelial growth and conidiophores of A. fumigatus viewed under a compound microscope.
Among the microparticles suspended in air, there is an abundance of biological material, including fungal spores, pollen grains, bacteria, and viruses. Air-conditioning systems can readily become polluted by these biological contaminants, which disperse throughout indoor areas and raise the risk of infection among the occupants [48]. The amounts of bacteria and fungi harbored by these systems tend to differ according to location, and numbers and frequencies also show seasonal differences [49]. Furthermore, differences in the number of microorganisms isolated and the distribution of different types of isolates can also depend on the type of filter used and the frequency of cleaning. In this regard, [20] found Cladosporium sp. to be a dominant contaminant and also identified Alternaria sp., Aspergillus flavus, Aspergillus niger, and Rhizoctonia sp. with frequencies of 24.16, 12.96, 12.8, 8.29, and 4.96%, respectively. Similar results were obtained by [50, 51, 52, 53]. Consistently, Al-Suwaine et al. [54] mentioned that Aspergillus and Cladosporium spp. were the common isolates detected in closed systems in Riyadh, KSA, whereas other fungal genera, including Fusarium and Rhizopus, were isolated in low frequency, similar to findings of [51, 55].
\nHeating, ventilating, and air-conditioning (HVAC) systems function by drawing in air through a network of intake ducts, cooling the air, and then releasing the cooled air back into the home through return ducts. The constant recirculation of air in HVAC systems means that pollutants are continuously blown through indoor areas. In the eastern region of Saudi Arabia, given that the summer is generally very hot and humid, many properties are at risk from the growth of mold within air ducts.
\nWe have previously examined the nature of the relationship between filters and airborne microbes, using small pieces (1 × 1 cm) of traditional filters (sponge, polyester, and HEPA) These materials were sterilized with alcohol, then dried, and subsequently moistened with glucose yeast extract medium. We then prepared suspensions of the examined fungal strains (Aspergillus niger, Aspergillus flavus, Cladosporium sp., and Rhizoctonia sp.). These were retained in sterilized petri dishes, whereas other groups were prepared for carbon-free sources. They were monitored for 1 to 3 months and thereafter examined under a microscope. Heavy growth of mycelium was observed. The fungal filaments are looped around filter fibers also assembled into the filter cavities, forming a tangled knot of fungal mycelium and filters fibers (Figure 11).
\nStructure of filters observed under an optical compound microscope. The right-hand panel shows sponge filter cavities of different sizes. These openings are wider than those of other filters. The middle panel shows a polyester filter, which is characterized as a network installation with narrower openings than the sponge filter that are regular in shape. The left-hand panel shows a HEPA filter, characterized by complex knit and numerous narrow openings that increase efficiency by preventing the passage of fine particles.
\nFigures 12–15 show that the microscopic structures of the filters shown in Figure 11 have been colonized by Aspergillus niger in (Figure 12), Aspergillus flavus in (Figure 13), Rhizoctonia in (Figure 14) and Cladosporium in (Figure 15) which indicate that these fungi use the filters as a support for fungal mycelium.
\nThe mycelial growth and conidiophores of Aspergillus niger on the studied filters: (a) sponge, (b) polyester, and (c) HEPA.
The mycelial growth and conidiophores of Aspergillus flavus on the studied filters: (a) sponge, (b) polyester, and (c) HEPA.
The mycelial growth of Rhizoctonia sp. on the studied filters: (a) sponge, (b) polyester, and (c) HEPA.
The mycelial growth and conidia of Cladosporium sp\n.\n on the studied filters: (a) sponge, (b) polyester, and (c) HEPA.
Microorganisms can exploit various parts of air-conditioning systems, including filters, as sheltered sites, which are conducive to rapid grow and reproduction [16, 17, 56]. The high levels of humidity in air-conditioning systems [16, 57] and the accumulated dust in the filters and other parts of these systems provide an environment that is suitable for the growth of a range of different microbes.
\nMicroorganisms can secrete a diverse array of extracellular enzymes to exploit the various available filter materials, such as cellulose, as sources of nutrition [56, 58].
\nKuehn [57] pointed out that moisture promotes fungal growth in filter tissues and can also favor bacterial reproduction leading to subsequent transmission to and dispersal within indoor environments. Such moisture often originates from the drops of condensate that form air-conditioning towers [59].
\nMaus [60] have suggested that the spores of some bacteria and fungi trapped within air filters can retain their viability and reproduce under the prevailing environmental conditions. These microorganisms can be dispersed through purification and air-conditioning systems and be inhaled by workers and residents in buildings [48].
\nMicrobiological particles constitute one of the most important sources of air pollution that determine the purity of the air. It is known that atmospheric air is a carrier of disease-causing organisms, including fungal spores and microbial cells, the concentrations of which vary widely according to environmental condition, particularly the nature of the internal environment and the various activities of humans who reside or work therein [61]. In order to achieve the desired level of microbial contamination control in air-conditioning systems and develop suitable air purification techniques, it is generally necessary to conduct extensive studies [17].
\nHamada and Fujita [62] noted that the contamination of filters tends to be very low during the first year after installation, reaching 257 cells/m3 of room air, whereas by the sixth year of use, they found that the number of contaminating cells had increase threefold to 692.
\nDurand [63] demonstrated that species of Aspergillus and Penicillium are the fungi most commonly isolated from the filters of air-conditioning systems, whereas species of Cladosporium and other types tend to be detected at relatively low rates. For bacteria, species of Actinomycetes and Bacillus (cocci and Gram-negative types) tend to be the most commonly isolated.
\nAl-Abkari [17] recommended that air-conditioning filters should be cleaned regularly and that regular maintenance is necessary to prevent an accumulation of contaminants and to remove the suspended dust. In this regard, spongy filters can be readily washed and cleaned with detergents, and be reused after cleaning. In contrast, HEPA filters, which consist of interlocking fibers, are very difficult to wash and clean and should thus be replaced on a regular basis. Furthermore, it has been demonstrated that the number of microbial colonies (bacteria and fungi) growing on culture dishes that were exposed to air that had passed through different filters increased after 30 min and then decreased after 60 min. This indicates an inverse relationship between the period of operation of the air-conditioning system and the quantity of air that had been purified [64].
\nAl-Abkari [17] examined the extent of microbial growth on the most common types of filter used in the eastern region of Saudi Arabia (sponge, polyester, and HEPA) and found that sponge filters harbored the highest microbial moist mass, reaching 0.177 and 0.257 gm for bacteria and fungi, respectively. Comparatively, the bacterial mass recorded on polyester and HEPA reached 0.024 and 0.037 gm, respectively, and the corresponding fungal mass on these filters was 0.072 and 0.047 gm, respectively. Douwes [65] isolated polysaccharide compounds known to be excreted by various fungi that grow on dust in residential homes, and the detection of these compounds is accordingly considered to be a good indicator of the presence of these fungi.
\nMoray and Williams [66] performed direct microscopic observations of the porous soft filters typically used in air-conditioning systems and accordingly identified pollen grains, cellulose fibers, synthetic fibers, decayed plant leaves, hairs, parts of insects, dust, mites, and numerous organic compounds, all of which can provide a refuge for microbes.
\nMicroorganisms that are captured by filters can thrive on the filters and can potentially be released into the air, thereby resulting in sick building syndrome [67]. Furthermore, it has been determined that the number of microbes found in indoor air is less than that colonizing the surface of filters used in air-conditioning systems [68]. Foarde [69] and Kowalski [68] examined the efficiency of these systems and provided solutions for the HEPA filters. In addition, they noted that the tested filter samples trapped Bacillus subtilis, with efficiencies ranging from 19 to 100%, whereas in contrast the efficiency in trapping viruses was low, ranging from 0.7 to 20%.
\nAl-Abkari [17] examined the ability of microorganisms (bacteria and fungi) to degrade various types of filter commonly used in air-conditioning systems, namely, sponge, polyester, HEPA, and the environmental conditions, such as dust, temperature, and moisture, which enable these organisms to take refuge, grow, and reproduce. The results indicated that the growth of bacterial strains was dependent on the filter media containing a carbon source. The average of bacterial moist mass loading on different filters was found to be positively related to the length of incubation period (1, 2, and 3 months), with weights reaching 0.061, 0.09, and 0.101 g after incubations for 1, 2, and 3 months, respectively. Furthermore, it was found that the average microbial mass detected on sponge filters (0.177 g) was larger than that on either polyester (0.024 g) or HEPA (0.037 g).
\nGenerally, it was observed that the average of moist weights of bacterial mass on all filters increases with an increase in the length of the incubation period, with recorded averages of (0.134, 0.169, and 0.228 g) and (0.019, 0.024, and 0.03 g) and (0.031, 0.035, and 0.046 g) for sponge, polyester, and HEPA filters, respectively. In contrast, it was found that the moist mass of microbial growth on culture medium lacking a carbon source remained essentially constant with increasing incubation time, with values of 0.023, 0.023, and 0.028 g; 0.03, 0.035, and 0.039 g; and 0.163, 0.171, and 0.162 g) for sponge, polyester, and HEPA filters, respectively.
\nWith regard to the growth of fungal strains, when these were grown in a medium containing a carbon source, the average moist fungal mass loading on different filters showed a positive relationship with incubation period (1, 2, and 3 months), with weights reaching 0.87, 0.118, and 0.142 g, respectively. Similar to bacterial growth, the average weight of fungal biomass growing on sponge filters (0.257 g) was larger than that on polyester (0.072 g) and HEPA (0.047 g). The weight of fungal mass on polyester and HEPA filters was 0.05, 0.082, and 0.085 g and 0.022, 0.04, and 0.078 g, respectively. Notably, however, fungal growth on sponge filters increased with increasing incubation time, reaching 0.181 and 0.324 g, following incubation for 1 and 2 months, respectively, whereas after incubation for 3 months, it had decreased to 0.265 g.
\nMicrobial pollution is one of the most fundamental indoor environmental quality problems in buildings. Therefore, this chapter has presented several solutions for indoor air quality monitoring in an effort to enhance the healthcare by describing the potential impact of HVAC systems on the indoor air quality. The principles of air filtration and traditional air filter types were presented.
\nAlso, the chapter covered the filtration technology and the indoor air quality topics. Subsequently, the air duct cleaning devices, mold formation, and HVAC systems and indoor pollution were illustrated. Moreover, this chapter provided the ASHRAE standards, which was used to select the suitable HVAC filters. The six most common designs of HVAC filters were briefly described. This chapter was followed by the modern filters. All advanced air filters, UV lights, activated carbon, deodorizing, antibacterial, electrostatic and HEPA filters, microbial filtration efficiency of HEPA filters were discussed extensively.
\nA brief description of the microbial colonization on the commonly used traditional filter types for air-conditioning systems were provided, followed by a detailed explanation of the relationship between traditional filters and microbe’s formation.
\nEven though there is highly development in designing advanced filters to overcome microbial pollution, we are still facing indoor air pollution problems. The most challenging step is providing an affordable construction, easy to install, made of environmentally friendly and long-term materials, available in different designs and able to avoid the existence of microbial growth.
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