**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.

**Keywords:** vitreocontrast, macular holes, vitreous anatomy, macular surgery, body visualization technique

#### **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 [1–7]. 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 [7–9]. 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 [4–6, 11–15].

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]:


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

*Perspective Chapter: The Vitreous Body Visualization Technique in Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.109264*

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 chromovitrectomy. 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 [27–30].

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, 31–33].

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, needleholders, 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**).

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**)

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 *Perspective Chapter: The Vitreous Body Visualization Technique in Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.109264*

#### **Figure 1.**

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

#### **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.*

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.

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

**Figure 3.**

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

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**).

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**)

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.

*Perspective Chapter: The Vitreous Body Visualization Technique in Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.109264*

#### **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.*

#### **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.*

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.
