Perspective Chapter: The Vitreous Body Visualization Technique in Diagnosis and the Classification of Idiopathic Macular Holes

Abstract

Application of the developed technology of vitreous body visualization in patients with macular holes (MH) of different sizes provides a principally new approach to clinical and diagnostic examination based on the development (depending on the stage of the pathological process) of basic classification anatomical and morphological signs (elongation of the vitreous cisterns in anteroposterior direction, violation of the integrity of the vitreous cistern wall and probability of staining composition exit beyond stained cavities, the degree of adhesion of the vitreous body on the internal limiting surface, etc). and characterized by advantages over traditional MH classifications, which in general (based on the outlined recommendations) makes it possible to significantly increase the clinical effectiveness of vitreoretinal surgical intervention in comparison with traditional classifications.

1. Introduction

Currently, researchers show big interest to the study of vitreoretinal interface, its components, adhesion mechanisms, processes leading to their disorder, and subsequent anatomic functional changes. Up till now, the role of cortical vitreous and retinal vitreous layers interrelation in the aspect of pathogenesis and full classification of idiopathic macular holes has been insufficiently disclosed [1, 2, 3, 4, 5, 6, 7]. The development of the new ways of intravital imaging of the structural components of the vitreous body and vitreoretinal interface is an important tool to improve clinicians’ knowledge of the pathogenesis of various vitreoretinal diseases in order to improve the methods and quality of treatment [7, 8, 9]. New staining solutions provide an opportunity to study anatomic topographic changes in the structures of the vitreous body (VB) and vitreoretinal interface (VRI) during surgical intervention with possible subsequent histological and ultrastructural studies [1, 10]. All this contributes to the development of innovative methods and modernization of existing approaches for the treatment of diseases of the posterior segment of the eyeball [4, 5, 6, 11, 12, 13, 14, 15].

The aim of this work was to study the anatomical and topographic specifics of changes in the vitreous body and vitreoretinal interface at different stages of idiopathic macular holes (MH). We followed up 143 patients (143 eyes), 105 females, 38 males, aged 51–78 years (mean age 64.8 ± 5.3 years). Inclusion criteria for patients were the presence of MH, transparent optical medium, the absence of diabetes mellitus, and other systemic diseases and ophthalmic surgical procedures in the medical history. The study exclusion criteria for patients were high myopia and traumatic MH. All the patients were divided into the following three groups according to the traditional classification of the International Vitreomacular Traction Study Group (2013) [13]:

• patients with small (≤ 250 μm) diameter MH (40 patients, 40 eyes);

• patients with MH of medium (>250—≤400 μm) diameter (58 patients, 58 eyes);

• patients with large (>400 μm) diameter MH (45 patients, 45 eyes).

At the same time (according to the classification), in all the cases this was a primary MH without vitreomacular traction .

Vitreoretinal intervention was performed using vitreocontrastography technique, a distinctive feature of which was the use of Vitreocontrast suspension to highlight VB and VRI structures in order to study their topographic anatomy [1, 2].

The VRI topographic anatomy was assessed and intraoperatively video recorded.

When cortical layer remnants were visualized on the retinal ILM surface, their topography, area, and configuration were assessed followed by photo and video recording of changes in VRI anatomy and determination of the borders of abnormal posterior vitreous detachment (PVD).

Application of the developed technology of vitreous body visualization in patients with macular holes (MH) of different sizes provides a principally new approach to clinical and diagnostic examination based on the development (depending on the stage of the pathological process) of basic classification anatomical and morphological signs (elongation of the vitreous cisterns in anteroposterior direction, violation of the integrity of the vitreous cistern wall and probability of staining composition exit beyond stained cavities, the degree of adhesion of the vitreous body on the internal limiting surface, etc.) and characterized by advantages over traditional MH classifications, which in general (based on the outlined recommendations) makes it possible to significantly increase the clinical effectiveness of vitreoretinal surgical intervention in comparison with traditional classifications.

2. Experimental study 1

Currently, glucocorticosteriod (GC) (triamcinalone acetonide (TA), Kenalog-40) suspensions are widely used as contrast staining agents for the structures of the vitreous body [8, 12, 16, 17]. In ophthalmology, these substances have been used since 1950s to suppress intraocular pressure and the proliferation of fibroblasts. In 1974, R. O. Graham described their intravitreal use for the treatment of experimentally induced endophthalmitis [18]. In 1988, Machemer R. confirmed the effectiveness of the intravitreal introduction of GC crystal forms for the local suppression of intraocular inflammatory and proliferative processes [16, 17]. Currently, GC is used to inhibit inflammatory processes in diabetic macular edema and cystic macular edema caused by the central retinal vein occlusion, subretinal neovascularization, and other vitreoretinal diseases. However, these medicine remedies may have such side effects as lens opacification, elevated intraocular pressure, and endophthalmitis [19].

In 2000, Peyman G. for the first time described TA as a vital dye for chromo-vitrectomy. During the study, it was found that the particles of the vital dye penetrate between the vitreous cortex fibers staining them, thus enabling their better visualization and facilitating the removal of these tissues [12]. Besides, not being a true dye but a suspension it settles on the VB in the form of precipitates, thus making it possible to easily distinguish VB from surrounding intraocular structures [20]. The staining agent precipitates on the surface of epiretinal membranes (ERMs) facilitating their subsequent removal [12, 21]. Clinical studies confirmed TA efficacy as a staining contrast agent for intraocular structures. Matsumoto H. et al proved that the removal of vitreous posterior cortical layers during vitrectomy is safer with the use of TA than without the use of the vital dye [22]. Enaida H. et al studied 177 eyes of 158 patients who had chromo-vitrectomy for rematogeneous retinal detachment, macular hole, proliferative diabetic retinopathy with TA and without it. The authors did not find any difference in the visual acuity in both groups. However, the number of retinal detachment recurrences that required repeated surgery was lower in the group where vitrectomy was performed with the use of TA [23]. The authors did not find any statistically significant difference in the occurrence of complications after the surgical intervention with the use of TA and without it [17]. Horio N., Furino et al. used TA to facilitate the removal of ILM and epiretinal membranes during vitrectomy. In all the cases, there was visual acuity improvement and no complications in the postoperative period [20, 21]. Nevertheless, some authors mention difficulties in ILM removal when TA is used as a staining agent. Due to the size of its particles, the dye spreads over the retinal surface making the macular tissue invisible, the manipulation is poorly controlled, and the risk of iatrogenic retinal damage increases [4]. The TA injectable form present at the domestic pharmacological market is Kenalog-40 suspension (Bristol-Myers Squibb., Italy). A dose of the drug for intravitreal injection contains 4 mg of TA active substance in 0.1 ml of solution. The suspension contains 40 mg of TA and 9.9 mg of benzyl alcohol in isotonic sodium chloride solution.

According to a number of authors, it is the benzyl alcohol contained in Kenalog-40 suspension that can have a toxic effect on the retina. The preservative contained in the suspension can cause necrosis of retinal pigment epithelial cells (PECs) and have a damaging effect on glial cells as opposed to TA in physiological solution. The toxic effect depends on the dose and exposure time of the solution. The TA crystals themselves have no cytotoxic effect on PEC cells. Nevertheless, Narayanan R. et al. refuted the data that the preservative contained in the suspension Kenalog-40 (Bristol Meyers Squibb., USA) has a toxic effect on retinal cells [24]. The TA and benzyl alcohol toxic effect on the structures of the posterior segment of the eye has been studied in the experiment on animals. The results of these studies are also contradictory. A number of researchers did not reveal the damaging effect of TA on the retina of rabbits [5, 25], while others demonstrated the toxic effect of the main substance and the preservative [24, 26]. Therefore, in order to avoid morphofunctional changes in the posterior segment structures during vitreoretinal intervention, it is recommended to use TA solution without a preservative, and the exposure time of the solution should not exceed 5 min. Thus, the use of TA facilitates the removal of VB and VRI structures during chromo-vitrectomy. However, this suspension has a number of side effects and can have a toxic effect on the structures of the posterior segment of the eye. The undeniable advantages and effectiveness of chromo-vitrectomy technology make scientists continue their research in this area.

At the S. Fedorov Eye Microsurgery Complex of the Russian Ministry of Health, Vitreocontrast suspension has been used for the intraoperative visualization of the vitreous body structures and the VRI since 2009. This staining agent (TU No. 9398-017-29039336-2009) is an ultradisperse suspension based on barium sulfate, insoluble in water and physiological liquids, a neutral nontoxic inorganic salt in an isotonic solution with osmolarity of 300–350 mOsm. Barium sulfate is a white crystalline substance with a molecular weight of 233.43 g/mol, particle size in Vitreocontrast suspension less than 5 μm, and density of 4.4 g/cm³ [27, 28, 29, 30].

Each 1.0 ml of sterile solution contains 140 mg of dry substance (barium sulfate) [6, 13, 15]. Various experimental studies have confirmed the safety of intraocular administration of the suspension [6, 13, 15]. Vitreocontrast is currently used to stain VB and VRI for retinal detachments, proliferative diabetic retinopathy, macular holes, and idiopathic epiretinal membranes.

Thus, both vital dyes have similar physical and chemical characteristics. They are biologically inert substances used to stain intraocular structures in different vitreoretinal pathologies. In order to choose the optimum vital dye, we carried out experimental staining of VRI by suspensions «Kenalog-40» and «Vitreocontrast», which made it possible to assess their main characteristics and to choose intraoperative staining of the eye posterior segment, to evaluate the results of the comparative staining by suspensions Kenalog-40 and Vitreocontrast in the PVD induction during the experimental study [27, 31, 32, 33].

The study was performed on 20 cadaveric human eyes. Kenalog-40 glucocorticoid suspension and Vitreocontrast suspension were chosen as VB staining agents. The staining ability of the proposed compositions was assessed on the VB macro preparation. A set of microsurgical ophthalmological instruments including: scissors, needle-holders, eye microsurgical forceps, ophthalmosurgical knives, insulin syringes, 27 G, 30 G needles, 25 G ports (Alcon, USA), and 25 G endovitreal forceps (Alcon, USA) were used to prepare eyeball and VB. In all the groups, non-fixed eyeballs were dissected in several stages according to the suggested original technology. Initially, the sclera was incised with scissors 4 mm from the limbus in a circle, leaving the anterior segment of the eye intact (cornea, iris, and lens). Then the sclera was cut between the rectus muscles not reaching the projection of the yellow spot and the place of optic disc exit forming scleral petals (Figure 1a and b).

Figure 1.

(a, b) Stages of preparation of eyeballs. a. Formation of scleral petals. b. Exposure of the choroid.

The scleral petals were cut off leaving a part of sclera with a dimeter from 10 to 11 mm in the posterior pole of the eye, which included the projection zone of the macula and the optic nerve. Then, with the help of the razor and the anatomical forceps, choroid petals were formed and also cut off (Figure 2a–c)

Figure 2.

The eyeball during the stages of preparation. a. Forming and cutting off scleral petals. b. Forming and cutting off choroid petals. c. Induction of posterior vitreous detachment by forming retinal petals and separating them from the vitreous.

The next step was to form retinal petals with the help of anatomical forceps, separate them from the VB surface leaving them fixed to the anterior segment and the posterior pole of the eyeball. Then the retina was separated from the VB surface, thus modeling the PVD. The VB and retina were examined to detect concomitant pathology.

Using a 25 G cannula and a 2.0 ml disposable syringe, 1.0 ml of Kenalog-40 suspension was applied on the vitreous surface and separated retina, then the surface was washed with saline to remove excess dye. The surface of VB and separated retina was photographed immediately after staining after 3, 5, 15, and 30 minutes. The staining intensity of the vitreoretinal interface structures was assessed visually. Then Vitreocontrast suspension was applied on the same area of the VB surface and separated retina, the surface was also washed, photographed, and the staining intensity was assessed visually. Next, the stained retinal sections and VB cortical layers were cut using Vanasse scissors, separated using scissors and anatomical forceps, and sent for histological examination. Morphological examination of stained VRI structures—retina and vitreous cortex—was done. For this purpose, the material was fixed in 10% neutral formalin solution, washed with running water, dehydrated in ascending alcohols, and embedded in paraffin. Then we made a series of histological sections using hematoxylin-eosin and Van Gieson staining and alcian blue staining. The preparations were studied under a Leica DMLB2 microscope at ×50, ×100, ×200, and ×400 magnification followed by photographing. Photographic registration was performed using a DFC-320 digital color camera included in the kit. After the instillation of glucocorticoid suspension Kenalog-40 on the retinal surface, not having sufficient degree of adhesion, the particles freely rolled off the surface of the retina and VB. In all the cases, the retinal surface remained visually unchanged, the vitreous surface was shiny smooth and completely transparent (Figure 3a–c). Repeated application of Kenalog-40 suspension on the surface of VB and retina did not change the results obtained earlier.

Figure 3.

The eyeball after instillation of Kenalog-40 suspension. a, b. The surface of the vitreous. c. The surface of separated retinal fragment.

The application of Vitreocontrast suspension on the vitreous surface and corresponding separated retinal lobes made it possible to visualize the sites of split cortical layers after experimental PVD induction. At these sites, a thin vitreous layer was detected on the retinal surface, and the vitreous surface corresponding to the retina was covered with a layer of Vitreocontrast suspension particles. At other sites, the vitreous surface remained smooth, shiny, and transparent, and the corresponding retinal sites also remained visually unchanged. The degree of suspension adhesion did not change over time, and vitreous fibers were clearly visualized on the retinal surface.

Thus, in case of true PVD and complete separation of VB from the retinal ILM, VB remained smooth, transparent, and shiny, Vitreocontrast particles did not adhere to its surface. In case of the tight adhesion of VB cortical layers to the retinal ILM during PVD induction, they split (vitreoschisis). In this case, the VB layer was visualized on the ILM surface (Figure 4a–d).

Figure 4.

The eyeball after the instillation of the Vitreocontrast suspension. Splitting of the cortical layers of the vitreous body during the induction of PVD. a and b. The surface of the vitreous body (black arrows). b and c. The retinal surface (red arrows).

The arrows indicate the vitreous fibers that were not contrasted during the instillation of Kenalog-40 suspension and which were contrasted after the instillation of the Vitreocontrast suspension.

The areas of the retina stained by Vitreocontrast suspension were excised and histologically examined to identify the stained layer.

On the histological preparation, cortical VB layers stained by Vitreocontrast suspension were visualized on the retinal surface of the donor eyeball. The retinal structure was unchanged. No visible artifactual damage was detected. There were signs of autolytic processes: moderate edema and loss of some nuclear layers, destruction of photoreceptor layer with elements of pigment granules (remnants of pigment epithelium), and fragmentary ILM detachment that were not aggravated by dispersed particles in the vitreal cavity (Figure 5a and b)

Figure 5.

Micro photography. Fragments of the vitreo-retinal interface, split cortical layers of the VB (black arowws) with adhered particles of Vitreoсontrast suspension (red arrows), the structure of the retina is unchanged.

The experimental study confirmed the possibility of vitreous cortical layers splitting in induced PVD. It should be noted that it was impossible to assess this condition when using Kenalog-40 suspension as a contrast staining agent. Not having sufficient degree of adhesion glucocorticoid suspension particles did not allow for the visualization of vitreous layer or the remnants of vitreous fibers on the retina. Thus, it was impossible to determine the areas of true PVD and the areas of vitreous stripping. On the contrary, the use of Vitreocontrast suspension as a contrast staining agent made it possible to visualize the areas of laminated vitreous cortex during PVD induction. The sites of the retina, on the surface of which the VB layer was stained, were sent for histological examination that confirmed the presence of the VB layer on the retinal tissue specimen after PVD induction.

The study revealed that during PVD, some cortical layers may completely detach from the retinal surface, while others, having a higher degree of fixation to the retinal surface, may split, thus forming an abnormal PVD. Today, Vitreocontrast suspension has a sufficient degree of adhesion and allows visualization of this condition—areas of true and abnormal PVD. Vitreocontrast has a number of advantageous properties. It stains VB structures in isolation, has high adhesion, due to which the intensity of staining of intravitreal structures does not change over time. Besides, it is a biologically inert substance. These characteristics indicate the feasibility of its introduction into ophthalmological practice in order to use it as a staining agent for VB structures and vitreoretinal interface during vitrectomy.

Comparative staining by Vitreocontrast and Kenalog-40 suspensions revealed that Vitreocontrast, due to its properties, determined by its physical and chemical characteristics did not stain only all intravitreal structures but also revealed the areas of cortical layer splitting in PVD. And the staining intensity of studied VB structures did not change with time.

The experimental study confirmed the possibility of splitting of cortical layers during PVD with the development of abnormal PVD. Under cortical layer splitting, a thin vitreous layer was visualized on the retinal surface, and the adhered particles of Vitreocontrast suspension remained on the corresponding laminated section of the vitreous.

3. Experimental study 2

The study was carried out on 40 cadaveric human eyes. In 14 cases, the vitreous body was stained by specialized contrast staining agent Vitreocontrast. The control group included the 14 eyes where Kenalog-40 suspension was instilled.

Simultaneous (comparative) contrast staining by Kenalog-40 + Vitreocontrast was performed in 12 cases.

Antegrade and retrograde dye injections were used.

In the control group, it was revealed that Kenalog-40 stains vitreous fibers but has a low-grade adhesion to the structural elements of the vitreous body. When Kenalog-40 was injected into the vitreous canals, it did not stay in their cavities. Vitreocontrast showed well-expressed adhesion to the VB structural elements (canals, cisterns, and their anastomoses), the degree of adhesion did not change with time. When injected in the projection area of VB canals, the dye settled on the walls of cisterns.

For the first time, a specialized contrast staining agent Vitreocontrast was developed, which due to its high mechanical adhesion, particle size, and intensive dye accumulation on VB structures made it possible to visualize not only VB fibers but also intravitreal channels, cisterns, and their relations.

One of the urgent issues of modern vitreoretinal surgery is intraoperative visualization of the vitreous body (VB), staining of its native structures and pathologically changed areas. The quality of the removal of transparent VB structures and the atraumatism of the intervention determine the anatomical and functional prognosis of the procedure. In this respect, the issue of VB structure undoubtedly remains important.

In 1973, J.Worst and Makhacheva Z.A. anatomical carried out functional studies of VB with the use of vital dyes that gave new understanding of its structure. Three rows of cisterns (the circle of equatorial, retrociliary and petaliform cisterns): canals (lentico-macular, optico-ciliary canal) and other VB structural elements were discovered and described [7]

Selection of perfect contrast staining agent still remains a topical problem due to the specific requirements (high dispersity, easy instillation and elimination, possibility of elimination via natural outflow ways, lack of side effects, etc.) imposed by surgeons.

Today, various vital dyes are used to identify the transparent structures of the posterior segment of the eye. These are triamcinolone acteonid (TA), trypan blue, [34, 35, 36] membrane blue (0.15%), fluorescein, and indocyanine green [15, 21, 23, 37]. At the same time, none of the vital dyes allows for the antemortem identification of intravitreal structures described in the works by J. Worst [7].

A number of researchers showed that modern vital dyes due to their nonselective diffusion stained not only VB but the surrounding intraocular structures too, which impaired the identification of VB. Besides, the abovementioned agents have a number of side effects (cytotoxic, phototoxic, cataractgenic action, elevated intraocular pressure, and pharmacological activity) [6, 11, 15, 16].

Staining by TA is more effective because being not a true vital dye but a suspension it settles on VB as precipitates and VB can be easily distinguished from surrounding intraocular structures [22, 23, 38, 39]. That is why we chose this agent to stain VB in the control group.

The purpose of this study is to develop a specialized contrast staining agent for the intraoperative vitreous body structures visualization.

The study was carried out on 20 cadaveric human eyes. Researchers used specialized agent Vitreocontrast and triamcinolone acteonid or kenalog-40 to stain vitreous structures.

Preliminarily, VB was completely dissected from the overlying membranes (conjunctiva, sclera, choroid, and retina) (Figures 6 and 7).

Figure 6.

The eyeball dissection. Sclera, choroid, and retina sequential removing.

Figure 7.

The eyeball after dissection.

In 14 cases, staining was performed by Vitreocontrast intravitreal injection.

The control group consisted of 14 eyes, in which kenalog-40 suspension was injected. Simultaneous (comparative) staining by Kenalog-40 + Vitreocontrast was performed in 12 cases.

Antegrade and retrograde methods of contrast staining agent injection were used.

We managed to stain the structures described in the works by J.Worst and Z.A. Makhacheva [7] staining VB structures with Vitreocontrast suspension retrograde injection. These were the circle of equatorial, petaliform and retrociliary cisterns, optociliary and lentico-macular canals, and premacular bursa (Figure 8a–c). Vitreocontrast showed pronounced adhesion to VB structural elements that did not change over time.

Figure 8.

(a-d) Vitreal body structures contrasting by the Vitreocontrast suspension (retrograde injection). Equatorial cysterns (red arrows) and lenticular cysterns (black arrows), retrociliary cisterns (green arrows) earlier J. Worst described.

The size of intravitreal structures was determined during the study (average size of retrociliary cisterns was 10–12 mm, equatorial cisterns were 15–17 mm, petal-like cisterns were 8–10 mm)

When staining with Kenalog-40 antegrade injection in the projection of lentico-macular canal was used the walls of the canal were not clearly visualized, the vital dye did not stay in the canal cavity gradually settling into the posterior hyaloid (Figure 9a–c).

Figure 9.

a. Kenalog-40 injection (anthegrade injection). b in the projection zone of lentico-macular the walls of the canal are not clearly visualized, the suspension settles in the VB hyaloid layers. c and d. Kenalog-40 particles settled on the vitreous cortical layers. The channels and ci sterns (their walls) not visualized. Retrograde administration of Kenalog-40 in the projection zone of lentico-macular canal, the walls of the canal are not clearly visualized, the suspension settles in the VB hyaloid layers.

Under the comparative contrast staining of VB structures, Vitreocontrast was injected in the lentico-macular canal in the macular projection after the administration of kenalog-40 suspension. Vitreocontrast stained this canal, anastomoses with the cisterns surrounding it, and the premacular bursa (Figure 9a–c).

For the first time, a specialized contrast staining agent Vitreocontrast was developed, which due to its high mechanical adhesion, particle size, and intensive dye accumulation on more loose structures made it possible to visualize not only VB fibers but also intravitreal canals, cisterns, and their relations (Figures 10–13).

Figure 10.

Vitreocontast suspension injection (red arrow) and Kenalog-40 injection (black arrow) in comparative vitreous structures contrasting.

Figure 11.

Vitreocontrast injection (red arrow) and Kenalog-40 (black arrow) in the comparative vitreous structures contrasting. The Vitreocontrast suspension provides retrociliar (red arrow) and equatorial cisterns (red arrow) contrasting.

Figure 12.

Vitreocontrast injection (red arrow) and Kenalog-40 (black arrow) in the comparative vitreous structures contrasting. The Vitreocontrast suspension provides retrociliar (red arrow) and equatorial cisterns (red arrow) contrasting.

Figure 13.

Vitreocontrast suspension injection (red arrows) and Kenalog-40 (black arrow) suspension in comparative vitreous structures contrasting.

The new contrast staining agent is not toxic that allows us to recommend it for the further use in diagnosis and treatment of vitreoretinal diseases after its clinical approbation.

4. Clinical study

4.1 Methods and results of the application of the developed technique of vitreous body visualization in the diagnosis of idiopathic macular holes

This aim of this work was to study the anatomical and topographic specifics of changes in VB and VRI at different stages of idiopathic macular holes (MH). We followed up 143 patients (143 eyes), 105 females, 38 males, aged 51–78 years (mean age 64.8 ± 5.3 years). Inclusion criteria for patients were the presence of MH, transparent optical medium, the absence of diabetes mellitus and other systemic diseases and ophthalmic surgical procedures in the medical history. The study exclusion criteria for patients were high myopia and traumatic MH. All patients were divided into the following three groups according to the traditional classification of the International Vitreomacular Traction Study Group (2013) [1]:

• patients with small (≤250 μm) diameter MH (40 patients, 40 eyes);

• patients with MH of medium (>250–≤400 μm) diameter (58 patients, 58 eyes);

• patients with large (>400 μm) diameter MH (45 patients, 45 eyes).

At the same time (according to the classification), in all the cases this was a primary MH without vitreomacular traction [33, 40].

The preoperative examination of patients was performed using traditional methods (visometry, tonometry; biomicroscopy of anterior segment, vitreous body and ocular fundus; B-scan, electroretinography and spectral optical coherence tomography (OCT). All the patients had a penetrating retinal defect in the macular area and increased (due to edema) retinal thickness along the MH edge. Besides, ocular fundus photography was made by MAIA fundus microperimeter (CenterVue, Italy) combining a black and white fundus camera with an image angle of 45° in addition to the perimeter. All the stages of the surgery including IIM peeling were recorded using a digital video camera Panasonic LQ-MD800E (Panasonic Corporation, Osaka, Japan) connected to operating microscope OMS-800 OFFISS (Topcon, Japan). Before each imaging session, routine exposure adjustment and calibration were performed by adjusting the white balance of the recording system to a standardized sample (Xpo-Balance, Lastolight Ltd., Coalville, Leicestershire, United Kingdom).

The intraoperative Vitreocontrastography was performed using regional infiltrational block anesthesia with central potentiation. Three-port vitrectomy was performed using 25 Short Totalplus Vitrectomy Pak under operating microscope Topcon OFFISS OMS 800 (Japan) on surgical machine Constellation (Alcon, USA). The ports were placed 4 mm from the limbus. BSS irrigation solution (Alcon Laboratories Inc., USA) was used to maintain the vitreous cavity volume during the procedure. Sterile air was used for postoperative tamponade of the vitreous cavity. All the patients underwent standard three-port 25G closed vitrectomy, a distinctive feature of which was the use of Vitreocontrast suspension for highlighting of VB and VRI structures in order to study their topographic anatomy. At the first stage, 3 25G ports were set at a distance of 4 mm from the limbus in the planar ciliary body projection at 2.30, 4.00, and 9.30 o’clock. Staining of the vitreous structures was performed sequentially through each port in order to visualize their preservation, size, topographic anatomy of intravitreal structures, and the degree of its destruction. In order to do this, we injected 0.1 ml in all the accessible quadrants using a 30G needle and stained the VB structures with Vitreocontrast suspension. After highlighting of intravitreal structures in all the segments and the video registration of anatomical topography of the VB condition, a three-port 25 G vitrectomy was performed using standard technique. The next stage was highlighting of the vitreous cortical layers. Then intraoperative induction of PVD and the removal of vitreous cortical layers with a vitreotome needle were performed in an aspiration mode. Then Vitreocontrast suspension was reapplied on the retinal surface, its excesses were removed by passive aspiration. The VRI topographic anatomy was assessed and video-recorded. If there were the remnants of cortical layers or vitreous fibers on the retinal ILM surface, their topography, area, and configuration were assessed. There were photo recording and video recording of changes in ILM topographic anatomy—abnormal PVD. In order to assess the degree of the adhesion of cortical layers to the retinal ILM surface, attempts to remove them by 25G endovitreal forceps were made. We also assessed the possibility of the cortical layer removal separately from the ILM, the degree of the adhesion, and the link of cortical layers to the MH edges. After the maximum possible removal of the vitreous cortical layers from the retinal surface, Vitreocontrast suspension was reapplied and the VRI topography was evaluated. After the mechanical removal of all the possible vitreous fibers, the retinal ILM was stained using 25G end-gripping Constellation Grieshaber forceps and the ILM around the MH was peeled. The proposed suspension made it possible to visualize the residual fibers on the ILM surface and to remove the ILM only within this area. As soon as the suspension settled on the ILM surface, the excess agent was immediately removed by passive aspiration. After the visual inspection of the macular area, we proceeded to the formation of ILM fragments using 25G end-gripping forceps. The formation of an ILM flap was started at 2.0–2.5 mm to the superior temporal arcade from the hole edge. Using micro forceps, an ILM section was separated from the retina by a pinch at the indicated point; then, gripping the ILM tip by forceps the membrane was cut off along 2–3 hourly meridians in a motion directed along the arc of an imaginary circle with MH in the center while making sure that there was no separation of the ILM from the hole edge. Then we intercepted the ILM separated along the arc at the end point and continued cutting off the ILM. By successive intercepting of the separated ILM section edges by forceps, we performed a circular separation of the ILM around the MH without the complete detachment of the fragment. Then we used a vitreotome in a “shave” mode to trim the edges of the dissected circular fragment of the ILM that were facing the vitreous cavity. After that, the ILM flap was placed in an inverted manner without mechanical impact on the foveola area.

The results of the study indicated that in the first group of patients in 64% of the cases the vitreous cisterns were elongated in the anteroposterior direction extending toward the posterior pole. The size of the cisterns was 6.5 mm in length; 8 mm in width; in 45% of cases there was disruption of the cistern wall integrity and the exit of the contrast composition outside the stained cavities that was determined from the viewpoint of VB destruction in general (64% of cases) (Figure 14).

Figure 14.

Visualization of intravitreal structures. The cisterns are elongated up to 10–12 mm, the contrast suspension out of the cavity can be seen (marked with an arrow), small MH, Group 1 patients.

After staining of cortical layers, intraoperative PVD induction with the presence of a Weiss ring was performed. At the current stage of research, this is a confirmation of the complete separation of cortical layers from retinal ILM. However, after the application of contrast suspension on retinal ILM in all the cases, a vitreous layer was highlighted on the retinal surface in the macular area that proved to the abnormal PVD and vitreoschisis (Figure 15).

Figure 15.

A vitreous layer on the retina after the PVD induction. On the retinal surface in the macular area, one can see a vitreous layer (vitreoschisis) with clear borders (small MH, I group patients).

Topographically, this vitreous layer is located in the center of the retina (macular area) and is of medium size, with 95% of cases characterized by a low degree of adhesion to the underlying tissues, friability and possibility of mechanical separation (Figure 16).

Figure 16.

Mechanical removal of the stained vitreous layer (small МH, I group patients).

Upon the repeated Vitreocontrast suspension application, the retinal ILM was visualized, but in 45% of the cases, no formed vitreous cortical layer was visualized on its surface. However, the ILM itself was visualized as a result of the adhesion of particles but as a thin “dusting” since the surface is rough. In 35% of the cases, residual vitreous fibers were visualized as separate areas, half of which could be difficult to remove mechanically.

In 45% of the cases, VB was visualized on the ILM surface in the central zone, but the layer was ultrathin. In cases when residual vitreous fibers or layers were present, we performed classical circular ILM peeling with obligatory capturing of pathologically changed ILM zones characterized by the presence of ILM areas with the tight adhesion of vitreous residues, which is a risk factor for ERM formation and proliferation.

The results of the study in the second group of patients evidenced that the destruction of the VB (changes in its shape, size, and integrity) was observed in 78% of the cases and was expressed in the elongation (up to 10 mm) of equatorial and retrociliary cisterns, which in 68% of cases was accompanied by the exit of the contrast agent from the cistern cavity and its settling in the macular projection zone. After staining of cortical layers and the intraoperative PVD induction, a suspension was applied on the retinal surface, and in 100% of cases, the vitreous layer was visualized on the ILM surface only in the macular area. No adhesion of the contrast suspension particles was observed throughout the rest of the retinal surface that indicated to the abnormal PVD with a vitreoschisis zone in the macular area. The vitreous layer visualized in the central zone of the retina (macular area) was of medium size. In 60% of the cases, it was characterized by a low degree of adhesion to the underlying tissues, friability and the possibility of mechanical separation (Figures 17–19).

Figure 17.

Macular hole contrasted.

Figure 18.

Contrasted macular hole area.

Figure 19.

The macular hole area contrasted can be defined and measured.

In the third group of patients, the VB destruction (changes in shape, size, and integrity) was observed in all the cases and was expressed in the elongation (up to 12 mm) of equatorial and retrociliary cisterns; in 95% of cases, it was accompanied by the exit of the contrast agent from cistern cavity and its settling in the macular projection zone (Figure 20).

Figure 20.

Visualization of changed intravitreal structures. Elongation of the part of cisterns in the anteroposterior direction (red arrow), complete destruction of the part of the cisterns (green arrow) exit of the contrast agent outside the cavity (blue arrow), big MH, III group patients.

The vitreous layer visualized in the center of the retina (macular area) is medium sized. In 15% of the cases, it was characterized by the low degree of adhesion to the underlying tissues, friability and the possibility of mechanical separation (Figure 21a and b and 22a and b).

Figure 21.

a. OCT patient pre-operation (patient А). b. Contrasting by Vitreocontrast. Vitreous body layer visualization in macular hole zone (patient А).

Figure 22.

a. OCT Patient B. b. Vitreous body visualisation in macular hole zone patient B.

Discussing the results presented, the following four main points should be mentioned. The first one is related to the choice of the basic classification of MH. In this regard, it should be noted that the concept, according to which the leading role in IMH pathogenesis is attributed to tangential vitreomacular traction (VMT), is currently generally accepted. The essence of the concept is that the radial vitreous fibers left on the perimacular surface after PVD are reduced, which gradually leads to a round-shaped retinal tear in the macular area. This theory was proposed by J. Gass [3]. And the classification of MH with distinguishing of 4 stages was developed on its basis. This classification is widely used at the present time. However, as far as the aim of this study is concerned, this classification has a significant disadvantage since it deals exclusively with the macular area. It does not address other elements of the disease pathogenesis (changes in the VB, vitreoretinal interface) outside the macular area [41, 42].

Currently, a new anatomical classification of MH ha has been created based on optical coherence tomography data [3, 43]. Research showed the sufficient efficiency of this classification in terms of typical OCT features at different stages of MH [6, 18, 26, 44], which in general determined the choice of this classification in the present study.

The second point determines the general aspects of MH pathogenesis from the standpoint of anatomical topographic changes in the vitreous. In this regard, it should be emphasized that the trigger for the MH development is the type of the abnormal PVD, which results in the adhesion of a vitreous cortical layer in the macular projection zone. This layer has a similar topography, area, configuration in all cases irrespective of the macular hole size. In small-diameter macular holes (as the initial stage of the pathological process), this layer has no pronounced adhesion to the underlying retinal ILM and in most cases can be removed from its surface. This fact accounts for the positive anatomical effect in small diameter MH in almost 90% of cases without performing retinal ILM peeling [12, 14, 43, 45]. Later, as the pathological proliferative process develops, the adhesion between the vitreous layer and the ILM increases and, apparently, the process is aggravated by the contractile abilities of the ERM in formation [46, 47]. This leads to the intensification of tangential tractions and the gradual growth of MH. This thesis was confirmed by a dense cortical layer that we discovered in the large-diameter MH group of patients. In the vast majority of cases, this layer is fixed to the retinal ILM. Thus, the leading role in the MH pathogenesis belongs to the changes in the ILM of the macular area that is the development of the vitreous layer adhesion in this region. In case of PVD, it leads to its abnormal development, and the residual vitreous cortical layer stays in the macular area on the ILM surface. As the pathological process develops, the degree of the cortical layer adhesion to the retinal ILM and the contractility and tangential traction increase, which leads to an increase in the size of the hole. It should be emphasized that in all the patients, regardless of MH size, anatomical topographic changes in the vitreous were identical and were expressed primarily in the development of destruction characterized by the elongation of equatorial and retrociliary cisterns and the exit of the contrast agent from cistern cavities and its settling in the macular projection zone. It is also important to note that the destruction of the vitreous was present in small-diameter MH, i.e., at the early stages of the disease, which may indicate that changes in the vitreous occur before the development of visible (ophthalmoscopic or OCT) or clinical manifestations. Changes in the topographic anatomy of intravitreal structures have no significant differences at different stages of idiopathic macular holes, hence do not significantly influence the progression of the pathological process. The third point is related to the results of our comparative analytical assessment of the clinical diagnostic efficiency of MH treatment according to the developed techniques of VB visualization and the traditional classification. Our analysis proved to the fundamentally higher level of anatomical and morphological diagnosis of the various stages of MH by the developed techniques of VB visualization (on the basis of the original Vitreocontrastography method). In our opinion, this is associated with the following general drawbacks in the traditional classification [3, 15, 45, 48]:

• according to a number of authors, the leading role in the development of MH belongs to changes in the vitreous, but even indirect data (including B-scanning) are not taken into account when making these classifications;

• in current classification, PVD is assessed only on the basis of OCT data, which is limited exclusively to the macular area;

• the main substrate for the development of the abnormal PVD is vitreous cortical layers with a tight adhesion to the retinal ILM. However, the existing classification does not allow assessing of the characteristic features (configuration, area, number, degree of adhesion to the ILM) of these cortical layers, which, according to some authors, are the main pathogenetic risk factor for the development of MH;

• there are virtually no data characterizing the occurrence and localization of vitreoschisis zones associated with vitreous lamination;

• the visualized band of the hyperreflective image is interpreted as HGM and PVD according to the classification, despite the principal impossibility of this method to visualize transparent structures and differentiate vitreous cortical layers;

• the development of pathological process on the retinal ILM surface around MH is generally considered to be based on the formation of the ERM that has tangential traction effect on the retinal ILM; however, ERM anatomical topographic characteristics are not provided in the classification.

Thus, the traditional classification of MH [1, 49] does not fully reflect the basic characteristics of the pathological process (volume, topography, degree of changes in the retinal tissue) and, most importantly, practically does not assess the changes in the VB, which can trigger this disease. The stated disadvantages are certainly related to the limitations of OCT data in terms of coverage, ability to visualize transparent structures, and dependence on technical capabilities of the equipment used. Practical application of the developed VB visualization technique (based on the original vitreocontrastography method) provided the following fundamentally new possibilities for anatomical and morphological evaluation of VRI under MH:

• visualization of changes in the vitreous structures involved in the pathological process;

• staining of vitreous cortical layers to control intraoperatively induced PVD;

• visualization of vitreous cortical layers adhered to the retinal ILM with the possibility to determine the exact size of vitreoschisis zone in any of the meridians;

• possibility to remove the visualized vitreous layer in order to determine not only the topographic anatomy but also to make histological preparations for light or electron microscopy or immunohistochemical studies;

• possibility to assess the number of vitreous fibers adhered to ILM surface after removing a vitreous layer and staining of the ILM surface;

• possibility to assess the necessity of ILM peeling.

The fourth point defines a number of MH classificational anatomic morphological signs (CAMS) from the standpoint of the vitreoretinal surgery improvement (Table 1).

№ п/п	Classificational anatomic morphological sign	MH size (according to IVTG classification (2013), [4, 22]
		Small (≤250 μм)	Medium (>250–≤ 400 μм)	Big (>400 μм)
1.	Removal of vitreous cisterns in anterior posterior direction	+	++	+++
2.	Disruption of the integrity of the VB cisterns wall and the exit of the contrast composition beyond the stained cavities.	+	++	+++
3.	VB destruction (violation of the integrity)	+	++	+++
4.	The degree of VB adhesion on the ILM surface	+	+++	+++
5.	The degree of VB adhesion in the macular area	+	+++	+++

Table 1.

Classification of MH anatomical morphological signs developed on the basis of the original technique of VB visualization.

Note: “−”—the sign is absent, “+”—the sign is insignificantly expressed; “++”—the sign is moderately expressed, “+++”—the sign is clearly expressed.

The data presented in Table 1 make it possible to formulate the following main directions of MH surgical treatment improvement provided by the VB visualization technique on the basis of the original vitreocontastography method:

• in the case of a small-diameter MH and the complete removal of cortical layers from the vitreous surface, it is possible to keep the ILM (patient 1); in the case of medium-diameter MH, the removal of IML can be performed either by the classical method that is a circular maculorrhexis with capturing of the entire ILM area with cortical layers adhered to it or by removing of the ILM with the formation of petals strictly along the contrast agent position keeping the ILM fixation along the hole edge and using any modification of the inverted flap (patient 2);

• in the case of a large-diameter MH and a bilayer vitreoschisis zone (or ERM), it is advisable to perform layer-by-layer removal with staining of each layer (CAMS - 3,4,5);

• in the case of large-diameter MH, it is advisable to perform maximum complete removal of the upper vitreous cortical layer keeping this layer fixed to the MH edges with subsequent staining of the underlying tissues. ILM with the vitreous layer is removed along the edge (or the area) of the stained ILM with the zone of the vitreous cortical layer adhered to it corresponding to the vitreoschisis zone (patient 3).

Thus, application of the developed technique of VB visualization (based on the original vitreocontrastography method) in patients with MH provides a principally new approach to clinical and diagnostic examination that is based on the development of basic classification anatomical morphological signs (visualization of structures and cortical layers (including on retina) of VB, the degree of VB adhesion, etc.) and characterized by principal advantages in comparison with traditional classifications of MR that in general makes it possible to significantly increase the clinical efficiency of vitreoretinal surgical procedures.

The points mentioned above were illustrated by the following clinical example.

Clinical case: patient Z., 64 years old, diagnosis medium diameter MH OS, at admission MCVA – 0.1, after the surgery MCVA – 0.3. The main stages of the diagnosis and treatment are presented in Figures 23–27.

Figure 23.

OCT data (preoperational examination), patient Z.

Figure 24.

a. Vitreous layer after the PVD induction, on the retinal surface in the macular area a vitreous layer with clear borders can be seen (patient Z. b. Abnormal PVD. On the retinal surface, a vitreous layer was visualized after complete intraoperative PVD induction (patient Z.). c. Removal of the cortical layer by forceps (patient Z). d–e. Removal of the cortical layer (patient Z.).

Figure 25.

(a and b) Vitreous layer was fixed to the edges of the MH (patients Z.). (c and d) Highlighting of retinal ILM after the removal of the vitreous cortical layer around the MH (patient Z.).

Figure 26.

(a and b) Removal of retinal ILM (patient z) (c) On ILM surface there was no vitreous layer (patient Z) (d) covering of the MH by the inverted ILM flap (patient Z).

Figure 27.

The flap stayed in the normal position during working with its fragments by the vitreotome (patient Z.).

5. Conclusion

Vitreoretinal pathology occupies a special place in the structure of eye diseases since these are the most complex and prevalent nosological forms that in many cases require highly qualified surgical treatment, and modern concepts of vitreoretinal surgery involve targeted, selective impact on the structures of the vitreoretinal interface. It should be also noted that the process of vitreoretinal intervention itself is constantly being improved in order to increase the clinical effeciency of surgical treatment both in the postoperative period and with regard to long-term results. The following main areas of improvement in vitrectomy can be conventionally identified as:

• the “technical" one aimed at developing and optimizing equipment for vitreoretinal surgical interventions;

• the “medicamental” one aimed at drug “support” for vitreoretinal surgery; the “diagnostic” one aimed at improving preoperative and intraoperative diagnosis

Currently, the following modern diagnostic methods that make it possible to visualize various vitreoretinal structures have been tested to the greatest extent. These are ultrasound examination (USE), optical coherence tomography (OCT), confocal laser scanning ophthalmoscopy (CLSO), and chromovitrectomy (CV). The use of water-soluble dyes and suspensions is considered the most relevant for the evaluation of intravitreal and preretinal structures within the framework of CV method. At the same time, despite the wide introduction of various staining agents for VB and retinal structures by CV technique into clinical practice, the selection of an optimal staining agent is still a topical issue due to specific requirements for vital dyes in terms of the visualization level as well as safety characteristics imposed on products that come into contact with the internal components of the eye for a long time.

However, in literature, there are only some sporadic studies that allow determining the size of intravitreal structures under normal, age-related, and pathological conditions of the vitreous and to define the comprehensive approach to the surgical treatment of vitreoretinal pathology depending on the morpho-functional condition. Thus, the problem of VB imaging needs further consideration both conceptually and along individual specific lines.

The abovementioned provisions served as a basis for the present study performed with the purpose of scientific substantiation, experimental and morphological development, and evaluation of the clinical efficiency of the complex technique of VB imaging for diagnosis and surgical treatment of vitreoretinal disorders.

The method of the study was based on three main conditions:

1. Complex approach to the assessment of the effeciency of the new VB imaging technique based on the study of anatomo-topographic, morpho-functional, clinical, sanitary-chemical, toxicological parameters as well as the patient’s “quality of life.”

2. Adequate and tested methods of collecting the required volume of clinical material (a total of 143 patients (143 eyes) and 6t autopsied eyes were examined).

3. Stage-wise approach (three stages) and the study consistency.

During the first stage of the study, the imaging efficiency and safety and various agents for chromo-vitrectomy were assessed on the basis of clinical and in vitro, ex vivo comparative assessment of various staining agents for the imaging technique of the vitreous body—, Kenalog-40, and original Vitreocontrastography method based on the use of Vitreocontrast suspension. Results of anatomical and morphological evaluation showed that Vitreocontrast due to its properties that are determined by its physical and chemical characteristics had a significantly higher level of visualization compared with Kenalog-40 as it stained not only all intravitreal structures but also the revealed PVD areas. At the same time, the researchers found that during PVD some cortical layers could completely detach from the retinal surface, while others having a higher degree of fixation to the retinal surface could laminate, thus forming an abnormal PVD. In this case, Vitreocontrast suspension had a sufficient degree of adhesion and allowed for the visualization of this condition.

Besides, it is important to emphasize that according to the studies, the Vitreocontrast suspension fully complied with the sanitary and chemical (pH 7.25 + 0.02 pH units (with the permissible value of 7.20–7.60 pH units). pH) and toxicological (according to the results of toxicity index (100 ± 10)% with the permissible values of 70–120%) indicators, the results of morphological evaluation (cyto- and phototoxicity in vitro), as well as the parameters of solubility (27 days), as well as parameters of adhesion resistance and biological inertness.

It is important to mention that in our opinion, the use of the new staining agent (Vitreocontrast) cannot be viewed from the point of chromovitrectomy improvement. The above principal advantages of Vitreocontrast suspension reasonably allowed us to substantiate a new, original technique of VB contrast staining that we called Vitreocontrastography (VRCG). The proposed term was adapted to vitreoretinal pathology based on the techniques widely used in medical practice (radiography, tomography, etc.) allowing for the determination of both qualitative and quantitative parameters of the organ under examination. The practical expediency of Vitreocontrastography technique application is explained by the following main advantages:

• high level of adhesion, due to which the intensity of staining of intravitreal structures does not change over time;

• possibility to stain not only all intravitreal structures but also the stripped cortex areas in PVD;

• possibility to determine middle-sized intravitreal structures;

• possibility to identify various anatomical variants of the arrangement of intravitreal canals;

• possibility to identify VB defects (hernias) as a pathogenetic risk factor for retinal detachment;

• required safety characteristics for products that come into contact with the internal structures of the eye for a long time in sanitary-chemical and toxicological (cyto- and phototoxicity) parameters, indicators of sterility, solubility, and biological inertness.

During the second stage of the current work, our efforts were dedicated to the development (in the ex vivo experiment) of the next algorithm (step by step) of macro and microscopic experimental morphological study of the anatomical topographic characteristics of isolated vitreous structures from the point of view of normal anatomy [28].

5.1 Macroscopic examination by the technique of preparation with staining of structures

1. Evaluation of anatomical topographic changes of vitreoretinal interface (by PVD to evaluate its view, whether it is true or abnormal).

2. Staining and evaluation of the topographic anatomy of intravitreal structures (canals, bursae, and cisterns), to evaluate the degree of VB destruction.

3. Evaluation of the topographic anatomy of the anterior cortex and vitreolenticular interface.

5.2 Microscopic examination by light (electronic) microscopy of each isolated VB structure

1. Isolated preparation of vitreous structures and layers.

2. Fixation by the original method with the use of a special substrate.

3. Evaluation of microscopic changes by light (electronic) microscopy.

5.3 Making an individual map of VB macromicroscopic topographic anatomy

This algorithm is characterized by the ability to distinguish any VB structure in isolation and to distinguish each cortical layer with the possibility to study its anatomo-topographic features and relations with the underlying tissues (retinal ILM, ciliary body, lens capsule).

The size of intravitreal structures was determined during the study (average size of retrociliary cisterns was 10–12 mm, equatorial cisterns were 15–17 mm, petal-like cisterns were 8–10 mm). Besides, it was revealed for the first time that the anterior cortical layers consistently line the pars plana, the posterior surface of the lens, the ciliary zone fibers and can laminate. Closer to the posterior surface of the lens, a tendency for gradual thinning and lamination of the detected VB layers came into being. For the first time, a separate anatomical structure in the Berger’s space projection zone was revealed, making us suggest the possibility of the existence of a retrolenticular bursa not previously described in literature, its anterior multilamellar wall was tightly fixed to the posterior lens capsule and inseparable from it mechanically. It is also important to mention that in the course of the preparation during the formation and cutting off retinal petals, cortex delamination was detected, which could serve as an adaptive mechanism to prevent retinal detachment. It was revealed by modern diagnostic techniques and interpreted as PVD. In addition, after the separation of cortical layers, a defect was formed in the area of the preoptic cistern or premacular bursa, with the subsequent separation of the posterior pole sclera section from the cortical layers and the development of vitreous herniation. In such a case, the VB structure was disturbed, Vitreocontrast suspension stains altered and elongated VB cisterns, and exited through the cortex defect. We noted that when the intraocular pressure increased, the cortex rupture occurred in the defect area with emptying of intravitreal structures and subsequent contraction of the cortical layers. This mechanism can serve as one of the links in the pathogenesis of regmatogenic retinal detachment.

Scientific substantiation of the complex technique of vitreous body imaging (CTVBI, original Vitreocontrastography method in combination with the developed step-by-step algorithm of macroscopic and microscopic examination) for the diagnosis and the surgical treatment of vitreoretinal pathology was based on the efficiency criteria that were developed using the following four groups of imaging methodological principles tested in literature:

1. Basic principles of information imaging in technical systems that made the basis of the following efficiency criteria—penetrating capacity, resolution, breadth of coverage, ability to apply digital data analysis, stability, subjectivity.

2. Main principles of intravital imaging in clinical anatomy that made the basis of the following criteria: possibility to determine the size of intravitreal structures, anatomic variants of the arrangement of intravitreal structures, prediction of pathological processes in retina from the viewpoint of defects in VB, and identification of disorders within an individual vitreous layer.

3. Main principles of morphological investigations in ophthalmology that made the basis for the following efficiency criteria: imaging efficiency in decreased transparency of optical media of the eye with a consideration of VB transparency in hard-to-reach localization or the minimal size of pathological changes as well as in the identification of normal and abnormal morpho-functional state of VB.

4. Main principles of creating the systems of vitreoretinal interface imaging tested in clinical practice including: versatility, staging, structured and stable conduction, realistic, controllable and segmentation of the obtained image with the possibility of pathological process modeling.

Comparative analytical evaluation of the developed technique with the approved methods of VB imaging according to the first three groups of efficiency criteria was performed in scores from 0 (no efficiency) to 3 (high efficiency). The results of the analytical evaluation revealed that the mean score of complex technique of VB imaging (CTVBI) was 2.8; CV -1.9; OCT - 1.4; ultrasound - 1.3 and CLSO - 1.2, respectively. Thus, the data obtained indicate the undoubted advantages of CTVBI where the level of VB imaging efficiency is 93% of the required one.

It is also important to note that our proposed system of the complex technique fully complies with the following basic principles of vitreoretinal interface imaging systems tested in clinical practice.

Universality principle was implemented in the possibility of imaging (based on the vitreocontrastography method) both in experimental (on donor eyes) and in real (intraoperatively during vitrectomy) conditions.

Principle of staging was characterized by our step-by-step technique of VB preparation.

Structuredness principle providing VB macroscopic and microscopic analysis from the position of the system of interconnected structures that in general allowed for the analysis of the internal structure and activating and regulating mechanisms.

Stability principle was characterized by the pronounced stability of the vitreocontrastography to adhesion throughout the surgical intervention.

Realism and controllability principles reflected the possibility of applying the developed VB imaging system in real time and in the required for each specific case duration and volume that is especially important in the process of surgical intervention.

Obtained image segmentation principle was characterized by the possibility of the isolated identification of each of the VB layers and structural elements (cisterns, canals).

Pathological process modeling principal lied in the effectiveness of the developed imaging system in detecting VB defects (hernias) as a pathogenetic risk factor of retinal detachment development.

The studies of the third stage of the work addressed the evaluation of clinical and diagnostic efficiency of the developed complex technology of VB imaging in MH.

The following anatomo-morphological classification criteria were determined for the diagnosis of MH of different sizes (143 patients, 143 eyes): elongation of VB cisterns in anteroposterior direction, disruption of the wall integrity of VB cisterns and exit of the staining composition beyond stained cavities, destruction (disruption of structural integrity) of VB, degree of VB adhesion to the ILM surface, degree of VB layer adhesion in the macular zone.

Our analysis testified to a fundamentally higher level of anatomical and morphological diagnosis of different stages of MH using the developed VB imaging technique that, in our opinion, was associated with the following general drawbacks of the traditional classification:

• according to a number of authors, the leading role in the development of MH belongs to VB changes; however, even indirect data (including B-scan) were not taken into account while developing this classification;

• in the current classification, PVD is evaluated only by OCT data that is restricted exclusively to the macular area;

• the main substrates of the development of abnormal PVD are VB cortical layers that are tightly adhered to the retinal ILM. However, the current classification does not allow the evaluation of the characteristic signs of such cortical layers (configuration, area, their number, the level of adhesion to ILM). And according to a number of authors, these cortical layers constitute the main pathogenic risk factor for MH development;

• there are practically no data characterizing the occurrence and localization of vitreoschisis zones associated with the lamination of the vitreous;

• the visualized band of the hyperreflective image is interpreted as PHM and PVD by the classification despite the principal impossibility of this method to visualize transparent structures and differentiate between vitreous cortical layers;

• according to the generally accepted views, the development of the pathological process on the retinal ILM surface around the MH is based on the formation of ERM that has a tangential traction effect on the retinal ILM, but the ERM anatomo-topographic characteristics are not provided for in the classification. Summarizing the above clinical data, it should be noted that in the present study the classification anatomo-morphological criteria established on the basis of the developed technique of vitreous body imaging allowed (taking into account the presented clinical examples) us to formulate a number of general and specific practical recommendations for the improvement of surgical treatment.

Thus, on the basis of the studies performed, theoretical provisions were developed. Accumulatively, they ensured an increase in clinical efficiency of vitreoretinal surgical intervention based on the application of the proposed complex system of VB imaging.

The practical application of the developed system of VB imaging is possible in the following areas:

• the research one—for macroscopic and microscopic (histological) examination of all the vitreous structures;

• the diagnostic one—for intravital imaging of the state and morpho-functional changes of VB;

• the surgical one—to increase the level of imaging during vitrectomy that ensures the high quality of surgical intervention.

6. Key take-aways

1. The results of experimental and morphological (in vitro, ex vivo) and clinical studies ensured the development of a comprehensive vitreoretinal imaging technique for the diagnosis and surgical treatment of various types of vitreoretinal pathology based on the use of original techniques (“Vitreocontrastography”, macro and microscopic vitreous body algorithm) as well as basic principles of vitreoretinal interface visualization (universality, stage-by-stage approach, structuredness, stability of carrying out, realism, manageability, and segmentation of received images with a possibility to model pathological process.

2. The developed complex technique of VB imaging is characterized, in comparison with traditional methods (ultrasound examination, optical coherence tomography, confocal laser scanning ophthalmoscopy, and chromovitrectomy), by significantly higher level of imaging efficiency according to three-point criteria developed in accordance with basic principles of intravital imaging in clinical anatomy (mean score – 3.0 0.5; 1.0; 0.25 and 1.5, respectively) and morphological studies in ophthalmology (mean scores of 3.0; 1.25; 0.75; 1.0 and 1.8, respectively).

3. The results of the complex (clinical, in vitro, ex vivo) comparative assessment of various staining techniques of vitreous body (MembraneBlue®Dual, Kenalog-40 (Triamcinolone acetonide), the original Vitreocontrastography technique) testify to the principal advantages of Vitreocontrastography associated with a high (93% of that required according to the developed criteria) level of imaging efficiency confirmed (according to digital colorimetry) by the highest (22.87 ± 6.67) value of mean Euclidean distance CIELAB), as well as compliance with sanitary and chemical (pH 7.25+0.02 units) values. pH (with an admissible value of 7.20–7.60 pH units), toxicological (by the results of the toxicity index evaluation (100 ± 10)% with admissible values of 70–120%) and morphological evaluation), solubility (27 days) parameters, resistance to adhesion and biological inertness.

4. An algorithm for macro- and microscopic experimental and morphological examination of the vitreous body was developed (in ex vivo experiment) that made it possible to determine the size of intravitreal structures (average size of retrociliary cisterns was 10–12 mm, equatorial—15–17 mm, petal—8–10 mm), the existence of a new structure (retrolenticular bursa), the mutual arrangement of the posterior zonules and anterior cortical layers and the three variants of PVD in terms of retinal detachment pathogenesis.

5. Application of the developed technique of vitreous body imaging in patients with macular holes (MH) of various sizes provides a principally new approach to clinical and diagnostic examination based on the development (depending on the stage of the pathological process) of basic classificational anatomical and morphological features (elongation of the vitreous cisterns in anteroposterior direction, violation of the integrity of the vitreous cistern wall and probability of staining composition exit beyond the stained cavities, the level of the vitreous adhesion on the inner limiting membrane surface, etc). and characterized by advantages in comparison with traditional MH classifications that allows for the significant increase in the level of vitreoretinal surgery clinical efficiency.

7. Practical recommendations

1. In order to improve surgical intervention for various types of vitreoretinal disorders, it is advisable to use the developed technique VB imaging based on the original Vitreocontrastography method providing (in general terms) the following advantages:

   • imaging of the changes in the vitreous structures involved in the pathological process;

   • staining of VB cortical layers to control induced PVD intraoperationally;

   • imaging of VB cortical layers adhered to retinal ILM with a possibility to determine the exact sizes of the vitreoschisis in any of the meridians;

   • the possibility to remove the visualized VB layer not only to determine the topographic anatomy but also to prepare histological preparations for light or electronic microscopy or immunohistochemical studies;

   • the possibility to assess the number of VB fibers adhered to ILM surface after VB layer removal and ILM surface staining.

2. Vitreoretinal surgical intervention (based on the developed technique) in patients with macular holes of different diameters should be performed taking into account the following recommendations:

   • In cases of the complete removal of VB cortical layers from ILM surface and the presence of small-diameter MH, it is possible to refrain from ILM removal or maculorhexis temporal side only (Figures 28–32)

Figure 28.

Cortical layers contrasting with Vitreocontrast suspension. Posterior vitreous detachment induction.

Figure 29.

ILM contrasting with remaining vitreous body on the surface.

Figure 30.

ILM pilling in the contrasted layer area, temporal side only, laying the lap on the projection of the macular hole. The layer is held in normal position due to Vitreocontrast particles adhesion.

Figure 31.

Vitreal tamponade by the air.

Figure 32.

OCT image of the patient (7 days after surgery).

ILM removal in the presence of a middle-sized MH can be done using the classical technique, i.e., circular maculorexis with the catchment of the entire ILM zone including the vitreous cortex adhered to it and the ILM removal with the formation of petals strictly according to the area of the staining agent location keeping the ILM fixation along the edge of the hole and with the inverted flap of any modification; in case of a bilayer vitreous zone in the presence of MH, a layer-by-layer removal with staining of each layer is advisable; in the presence of large-diameter MH, the maximum complete removal of the upper vitreous cortical layer is highly advisable (if possible) followed by staining of underlying tissues and preserving the fixation of this layer to the edges of the MH; In cases of the removal of the ILM with the cortical layer is performed according to the edge of the stained ILM with cortical layers adhered to it. The adhered zone corresponds to the vitreoschisis (Figures 33–39).

Figure 33.

Posterior vitreous detachment induction with Vitreocontrast suspension.

Figure 34.

Posterior vitreous detachment induction with Vitreocontrast suspension.

Figure 35.

ILM flaps forming (a–d).

Figure 36.

The reposition of inverted ILM flaps in the macular hole projection. ILM flaps held in the normal position due to Vitreocontrast properties.

Figure 37.

Cortical layer contrasting on the ILM surface. Cortical layer tightly bound to ILM had clear boundaries (blue arrows) and occupies a certain area that can be measured (black arrows).

Figure 38.

ILM flaps formation.

Figure 39.

Macular hole blocked by the inverted flap. ILM removed in the contrasted zone area, in this case ILM removed area corresponded with vitreoschisis zone after abnormal posterior vitreous detachment progress.