Histopathologic correlates of trabecular meshwork in microincisional trabeculectomy

Abstract

Purpose:

To report the histopathologic correlates of trabecular meshwork (TM) specimens procured by microincisional trabeculectomy (MIT) for different severities of glaucoma (early glaucoma: visual field mean deviation [MD] <−6 dB, moderate glaucoma: MD from − 6 to − 12 dB, and advanced glaucoma: MD <−12 dB).

Methods:

TM specimens from four patients undergoing MIT with or without cataract surgery were analyzed by routine histopathology for structural changes. The number of cells, the number of cells with spindle-shaped nuclei suggestive of epithelial–mesenchymal transformation (EMT), and the distance between the trabecular beams were calculated using different tools on freely available ImageJ software using the line or pint/count tool.

Results:

The TM specimens procured from two early and two advanced glaucoma cases showed decreasing cellularity and decreased compact arrangement of the trabecular beams in severe disease stages. The number of cells and preserved architecture in all four specimens were evident, with > 50 cells being present per section in all four cases despite the glaucoma being of advanced disease stage in two patients.

Conclusion:

The TM specimens obtained from MIT can be utilized for downstream analysis using different molecular methods for studying the molecular events in the tissue from early to severe glaucoma.

Trabecular meshwork (TM) represents the tissue of primary damage in any form of glaucoma.[1,2,3,4] Several molecular events precede the onset of structural or functional damage in the tissue resulting in manifest clinical features like raised intraocular pressure (IOP) and optic nerve/visual field damage. Decreased cellularity and loss of regular compact arrangement of the trabecular beams with epithelial–mesenchymal transition (EMT) changes are some structural changes that have been reported on histologic analysis, with majority of these studies being on cadaveric eyes with end-stage glaucoma.[3,4,5,6,7,8] Yet studying TM in vivo is challenging since the TM specimens obtained from routine trabeculectomy done in moderate–severe glaucoma contain very few viable cells, precluding useful tissue analysis by any molecular method like transcriptome or immunoblotting.[3,4,7,8,9,10,11] Early glaucoma patients are rarely offered surgery, keeping in mind the catastrophic complications associated with routine trabeculectomy, which makes it impossible to study the molecular events in the tissue in early disease stages. This mandates a study of in vitro models for studying disease pathogenesis, with clinical in vivo validation of in vitro being a challenge.[7] Frequent contamination of the sclera and corneal tissue in the dissected specimens with frequent absence of the TM tissue also adds bias to the results of any method of tissue analysis procured from routine trabeculectomy or other goniotomy procedures involving forceful cutting of TM, which destroys the natural architecture of the TM tissue.[11] Microincisional trabeculectomy (MIT) is a method of trabeculectomy with minimal complications, which achieves effective IOP control while ensuring a gentle removal of purely TM, without touching the cornea and sclera, through an ab interno approach.[9] We present the histopathologic correlates of TM obtained from MIT from four patients, including those with early–advanced glaucoma.

Methods

This study was a consecutive case series of four glaucomatous eyes undergoing MIT with or without cataract surgery between June 2022 and March 2023. Inclusion criteria included all patients with primary glaucoma with uncontrolled IOP >21 mmHg, progressive damage despite medications, or who were intolerant to medicines, with or without significant cataracts. Primary open-angle glaucoma (POAG) or primary closed-angle glaucoma (PACG) was diagnosed in patients aged >40 years with the above criteria. Juvenile open-angle glaucoma (JOAG) included patients aged 3–25 years with raised IOP, glaucomatous disc, and visual field damage. Pseudoexfoliation glaucoma (XFG) included those eyes with the above criteria with evident pseudoexfoliation (PXF) deposits. An informed written consent was obtained from all patients for this study which was approved by the institutional review board and followed the tenets of the Declaration of Helsinki. The clinical details including the visual field mean deviation (MD) and IOP at the time of surgery, along with the number of medicines were retrieved from the hospital database. Glaucoma severity was defined based on visual field MD as follows: early glaucoma MD <−6 dB, moderate glaucoma MD from − 6 to − 12 dB, and advanc ed glaucoma MD <−12 dB. The procedure for MIT is described elsewhere.[9] Briefly, the TM tissue was grasped with microforceps after a goniotomy incision with an Micro vitreoretinal (MVR) blade in the nasal quadrant followed by gentle stripping over 3–6 clock hours, which was collected in a filter paper and transferred in formalin for processing. Each specimen was then processed in formalin-fixed, paraffin-embedded tissue sections, which were stained with hematoxylin and eosin (H and E) and periodic acid–Schiff stain for staining the trabecular beams and cells. After H and E staining, each specimen was analyzed for the cell count per high-power field (HPF) and morphology using the point/count function on ImageJ (available on https://imagej.nih.gov/ij/download.html) with the scale reset to the specified slide dimensions (20×, 40×, or 100×) [Fig. 1]. ImageJ is a freely available online tool that allows objective evaluation of cell count and different characteristics of various images, avoiding subjective errors and is repeatable. The space between the trabecular beams was calculated using the line tool on ImageJ software to assess the compactness of the packing of TM beams [Fig. 1].

Figure 1.

The left panel shows the method of measuring the distance between trabecular beams using the line tool (arrow) on ImageJ, while the right panel shows the use of the point/count tool (arrow) on ImageJ for counting the number of cells

Case 1

This was a 49-year-old male with POAG with a baseline IOP of 36 mmHg mandating antiglaucoma medications. Table 1 gives the clinical details of all patients with the severity of disc and field damage at the time of surgery. Yet he was hypersensitive to most classes of available glaucoma drugs (save for pilocarpine), with IOP on medical possible treatment being around 28–30 mmHg on diurnal phasing. This mandated MIT for controlling IOP. TM removed from this patient revealed viable cells (134 cells per HPF) with intact trabecular beams which were tightly packed. Some TM cells were found to have assumed fibroblastic transformation (spindle-shaped nuclei), suggesting EMT [Fig. 2].

Table 1.

Clinical characteristics of patients undergoing MIT for harvesting trabecular meshwork

	Diagnosis	Age (years)/sex	Visual field MD/PSD (dB)	Baseline IOP (mmHg)	Surgery done	Clock hours of TM stripped
1	POAG early	49/Male	−3.5/2.66	36	MIT	3
2	JOAG	44/Female	−22.49/10.27	28	MIT	5
3	Advanced POAG	67/Male	−32.2/3.4	32	MIT with cataract	5
4	PXG	73/Male	−3.56/2.66	26	MIT with cataract	3

IOP=intraocular pressure, JOAG=juvenile open-angle glaucoma, MD=mean deviation, MIT=microincisional trabeculectomy, POAG=primary open-angle glaucoma, PSD=pattern standard deviation, PXG=pseudoexfoliation glaucoma, TM=trabecular meshwork

Figure 2.

(a–d) The structural changes in the trabecular meshwork (hematoxylin and eosin staining, 20 × or 40×) in four eyes that underwent MIT (see text for a detailed description) MIT = microincisional trabeculectomy

Case 2

This was a 44-year-old female with JOAG, uncontrolled IOP, and advanced disc damage despite the maximum tolerable medical therapy including systemic acetazolamide inhibitors. She underwent MIT for IOP control, which was subjected to histopathologic examination. The tissue specimen in this patient showed a majority of cells with fibroblastic transformation with loose trabecular beams, suggestive of loss of connective tissue typical of advanced disease [Fig. 2]. Yet the number of cells that were detected in the specimen was 54 per HPF, which suggested that viable cells are possible to be retrieved from the TM specimen even in the advanced disease stage [Table 2].

Table 2.

Analysis of the structural changes seen in TM surgical specimens procured from MIT

Number of cells per high-power fielda	Cells with spindle-shaped nucleia	Space between TM beams (µm)a
134	11	1.2–4.3
54	19	2.5–4.7
10	10	6–11
132	26	3.09–6.7

TM=trabecular meshwork. ^aSee text for a detailed description of the method for quantitative analysis using ImageJ

Case 3

This was a 67-year-old male with POAG who required concurrent cataract and glaucoma surgery owing to severe disc damage. The specimen collected showed sparse cells (10 cells/HPF), all of which were fibroblastic in nature with very loose trabecular beams [Fig. 2 and Table 2].

Case 4

This was a 73-year-old male with XFG with cataract and baseline IOP of 26 mmHg. The patient was unable to afford two long-term medicines given for IOP control and was found noncompliant over three visits with a visually significant cataract. He, therefore, underwent combined cataract and MIT surgery. The tissue specimen was found to have plenty of viable cells (132/HPF) with round nuclei, with very few cells with EMT changes. The TM beams were more tightly packed than the three cases, with < 10 cells with spindle-shaped nuclei.

Discussion

The TM tissues harvested by MIT showed potential structural changes that were reflective of the severity of damage in each form of glaucoma. Most importantly, even in advanced glaucoma, the TM architecture seemed to be preserved with identification of the trabecular beams and evident viable cells in the TM specimens, which makes this procedure suitable for analysis by immunohistochemistry (IHC), immunoblotting, proteomic or transcriptomic analysis. Loss of compactness of trabecular beams with decreased cellularity was evident with more intertrabecular spaces in advanced disease stages (cases 2 and 3) compared to early forms of glaucoma as seen in cases 1 and 4. The number of cells with EMT changes was also different between different severities of glaucoma, which concurs with previous histopathologic findings on TM.[1,3,4,5,6,7] A clear comparison of the sections between these TM tissues and those harvested using goniotomy reveals minimal trauma to the tissues by the surgery, thereby preserving the TM cells and making them more amenable for further research purposes.[11] While comparison with control or with different forms of glaucoma may not give concrete conclusions from this study, the utility of MIT for harvesting TM tissues with intact architecture and TM cells (unlike other minimally invasive procedures) amenable for downstream analysis is evident from this study. These can have a wide range of applications for basic research in glaucoma using in vivo tissues from different patients.

External TM specimens are variable in their results owing to lack of specific TM cell markers, frequent absence of TM in surgical specimens that contain sclera or cornea, and low count of viable cells (and therefore, DNA, RNA, or protein), which is also confounded by the presence of cornea and scleral tissues. This also makes it impossible to study cellular events in the TM surgical specimens harvested by external trabeculectomy, mandating in vitro models.[7] Since trabeculectomy is conventionally offered only in moderate–severe glaucoma, the study of the tissue-related events in early disease stages was impossible until the advent of microinvasive glaucoma surgery (MIGS). Different study groups have evaluated the utility of TM specimens harvested by trabectome, goniotomy blade, or TRabex with preserved architecture in the samples.[10,11,12] Yet morphologic analysis in an objective manner, as done with ImageJ in our study in different severities of glaucoma, was lacking in these studies. Further, a preliminary relook of the specimens in this study suggests that MIT helps preserve the architecture and TM cells better, as evidenced by a direct comparison of images used in other studies. Yet the difference in molecular analytical methods used on TM procured from different methods of MIGS is unclear at this point.

This may have a wide range of applications in clinical and basic research. It remains to be seen if this can be used to prognosticate glaucoma better or even predict markers for progression. This may help in altering and decision-making in clinical glaucoma practice by enhancing earlier detection of progression at the molecular level.

This study had several limitations. We only included early or advanced glaucoma, while we are still evaluating structure (on imaging)–structure (histopathologic) correlates in all stages of glaucoma using MIT. This may have a relative spectrum bias since we compare the mildest to the worst clinical scenarios. We did not compare these with controls, since procuring TM from an eye without glaucoma is still unethical, with the future possibly allowing TM “biopsy” from controls for comparative analysis, while ensuring safety and ethical concerns. Validation of TM-specific markers in the TM specimens from different types of glaucoma is still warranted to fully understand the histopathologic correlates of TM in glaucoma in a new manner that may reveal some new insights in contrast to earlier knowledge based on cadaveric eyes. Ongoing studies on larger sample sizes and different forms of glaucoma are mandated to validate the results obtained in this study. The application of this study will help understand the pathogenesis of TM in different stages and types of glaucoma in vivo and can also provide insights into the mechanism of damage.

Conclusion

Retrieval of TM specimens by MIT would have wide applications for studying TM-related mechanisms of disease pathogenesis in glaucoma.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship:

Nil.

Conflicts of interest:

There are no conflicts of interest.