Downregulation of BM88 After Optic Nerve Injury

Abstract

Purpose.: BM88 is a cell-cycle exit and neuronal differentiation protein that has been used as a marker of surviving retinal ganglion cells (RGCs) after optic nerve injury. Thy1.1 has also been used as a marker for RGC loss, but after optic nerve crush (ONC) a decrease in Thy1.1 expression precedes the loss of RGCs. The purpose of this study was to determine if BM88 expression was correlated with RGC loss after ONC and optic nerve transection (ONT) injuries.

Methods.: Rats were injected with Fluorogold (FG) into the superior colliculus to label RGCs and received ONC or ONT 7 days later. Eyes were collected 2 to 28 days after injury. Retinas were labeled with BM88 and intensity of the BM88 cell labeling was measured.

Results.: In control retinas, 98.9% of RGCs were immunoreactive (-IR) for BM88. There was a significant downregulation of BM88 by 52% to 80% of RGCs 7 days after ONC or ONT. The staining intensity of the remaining labeled cells was reduced to 41% to 51% of the control after 28 days of optic nerve injury. However, early in the injury there was a significant increase in the staining intensity of BM88.

Conclusions.: Nearly all BM88-IR RGCs colocalized with FG-labeled RGCs in control retinas. However, both the number of BM88-IR RGCs and their intensity decreased gradually between 4 and 28 days, preceding the loss of FG-labeled cells. These findings indicate that BM88 is not a good marker of surviving RGCs but may indicate abnormal RGC functioning, which precedes cell death.

Introduction

BM88 (C38 or Cend1 for cell-cycle exit and neuronal differentiation) is a protein that developmentally induces cell-cycle exit and neuronal differentiation. BM88 is an integral membrane protein that contains two 22 to 24 kDa polypeptide chains that are connected by disulphide bonds. ¹ It is anchored to the outer membrane of mitochondria and the endoplasmic reticulum, but may also exist in the cytoplasm. ¹ BM88 is expressed by retinal ganglion cells (RGCs) and horizontal cells in the retina (Fig. 1). ² BM88 overexpression in cultured cells results in cell-cycle arrest and silencing the BM88 gene accelerates cell proliferation. ³ BM88 forms a part of the p53-cyclin D1-pRb signaling pathway that leads to an end of the cell cycle at the G₀ phase. ³ BM88 induces p53 and p21, which inhibits cyclin D1 function. ³ Since cyclin D1 is inhibited, it can no longer phosphorylate pRb leading to cell cycle exit. Another reported role of BM88 in development is related to neuronal differentiation. Late in neurogenesis, BM88 acts on proneural genes, Mash1 and neurogenin1, to cause neuronal progenitors to differentiate into neuronal precursor cells and neurons. ³ It also releases Notch inhibition of proneural genes resulting in neuronal differentiation. ⁴  

Figure 1

 

Micrograph showing BM88 labeling in an uninjured retina (A). BM88 immunoreactivity was seen in RGCs, throughout the inner plexiform layer (IPL), in presumed amacrine cells (AC) in the proximal inner nuclear layer (INL), horizontal cells (HC) in the distal INL, throughout the outer plexiform layer (OPL), and intense labeling of the photoreceptor outer segments (Ph). Seven days after ONC the labeling of the retina was unremarkably different except that there were fewer RGCs labeled and their staining intensity was lower (B).

BM88 also plays a role in calcium buffering and therefore may have a neuroprotective activity. BM88 overexpression in neuroblastoma cells has resulted in neuroprotection against several neurotoxic stimuli. ³ BM88 inhibits proliferation of neurons by promoting the activity of the P2Y2 receptor and decreasing the activity of the P2Y1 receptor. ⁵ How it may achieve this is by blocking the release of calcium from inositol 1,4,5-triphosposte (IP₃) sensitive stores, such as in the endoplasmic reticulum, by either acting on IP₃ or the IP₃R. ⁵ This calcium-buffering activity prevents apoptosis by increasing the uptake of calcium by the mitochondria and lowering levels of calcium inside the endoplasmic reticulum. ⁵ Another antiapoptotic role for BM88 is its ability to inhibit extracellular signal regulated kinase (ERK). ⁵ When ERK is inhibited, Bcl-2 associated X protein (BAX) cannot bind to the mitochondria, which prevents apoptosis. 

The cell cycle of differentiated neurons must be kept under constant control because re-initiation of the cell cycle can lead to cell death. ³ In neurodegenerative diseases, there may be dysregulation of cell cycle arrest that may put neurons into an apoptosis-prone state. ³ There are examples of cell cycle protein reexpression in Alzheimer's disease, Parkinson's disease, ischemia, and amyotrophic lateral sclerosis. ^6–10 The importance of downregulating cyclin D1 in post mitotic neurons is emphasized by the observation that cyclin D1 reexpression is observed in neurons from patients with neurodegenerative diseases. ¹¹ Hence, BM88 may play an important role in the suppression of the cell cycle in mature neurons. 

BM88 has been used as a reliable marker of RGCs after optic nerve injury because its expression would not be expected to change because it is involved in maintaining cell-cycle arrest and RGCs are post mitotic. ^2,12,13 THYmocyte differentiation antigen 1.1 (Thy1.1) has also been used as a marker of RGCs to evaluate RGC loss after transection. ¹⁴ However, after optic nerve crush a decrease in Thy1.1 expression preceded the loss of RGCs. ^15,16 This suggests that there are differences in the expression of RGC markers depending on the type of injury. The purpose of this study was to determine if BM88 expression was correlated with RGC loss after optic nerve crush (ONC) and complete optic nerve transection (ONC) in young adult rats. 

Methods

Animals

Adult, female Sprague–Dawley rats (225–250 g; Charles River, Wilmington, MA) that were free of common pathogens were used in all experiments. Animals were cared for according to the guidelines of the Canadian Council on Animal Care. The animals were kept on a 12 hour light cycle and had access to food and water ad libitum. In all experiments the rats were anesthetized with an intraperitoneal injection of 7% chloral hydrate (0.42 g/kg of body weight; Thermo Fisher Scientific, Ottawa, ON) during experimental procedures. An ophthalmic eye lubricant (Lacri-Lube; Allergan, Markham, ON, Canada) was applied to the eyes before surgery. Animals were kept warm on a heating blanket (38°C). Animals were given subcutaneous injections of Anafen (5 mg/kg; Merial Canada, Baie D'Urfé, QC) to minimize discomfort following surgery. They were also given 5 mL of saline subcutaneously and allowed to recover on a heating blanket. 

Retrograde Labeling of Retinal Ganglion Cells

Retinal ganglion cells were retrogradely labeled before the optic nerve crush by injecting Fluorogold (FG; Fluorochrome LLC, Denver, CO) bilaterally into the superior colliculus as described by Koeberle and Ball. ¹⁷ The rat was placed on a stereotaxic frame (David Kopf M900; David Kopf Instruments, Tujunga, CA) and an incision was made on top of the head extending in the midline from a point 1 to 2 mm caudal to the eyes to a point 1 to 2 mm rostral to the ears. Once the incision was made, the transverse suture was located. A 1-mm hole was drilled bilaterally 2.5-mm rostral to lamda and 1.2-mm lateral to the sagittal sutures. Four microliters of FG was injected 3-mm deep into the parenchyma of the brain using a 10 μL Hamilton syringe (M701; Hamilton, Reno, NV) with the aid of a microinjector (WPI UltraMicroPump, Sarasota, FL). 

Optic Nerve Injury

Optic nerve crush or optic nerve transection (ONT) injury was done 1 week after FG injection. The animals were prepared for surgery as previously described. An incision was made in the skin around the rim of the orbital bone to access the optic nerve. The orbital contents were retracted away and the rectus muscle was moved laterally. The eye was rotated temporally to expose the optic nerve. The optic nerve was either crushed by compressing the intradural optic nerve for 3 seconds ¹⁸ using Dumont SS fine forceps (Fine Science Tools 11203-23; Fine Science Tools Inc., North Vancouver, BC), or transected by removing the optic nerve from the dural sheath and cutting the nerve as previously described. ¹⁷ A total of 53 animals were used in this study (post-ONC: day 0, n = 6; day 2, n = 4; day 4, n = 6; day 7, n = 6; day 14, n = 6; day 28, n = 4; post-ONT: day 2, n = 4; day 4, n = 4; day 7, n = 4; day 14, n = 4; day 28, n = 4). 

Tissue Preparation

The rats were euthanized between 2 and 28 days after injury or injection by injecting a lethal dose (5 mL) of 7% chloral hydrate. The eyes were enucleated and the cornea and lens were dissected away. The eyecup was fixed in 4% paraformaldehyde, 2% sucrose in Sorensen's phosphate buffer (0.1M, pH 7.3) for 2 hours. The eyecups were rinsed in sodium PBS (0.1 M, pH 7.3, 0.9% NaCl) 3 times for 30 minutes each. The eyecups were placed in 30% sucrose in PBS at 4°C overnight. The eyecups were embedded in OCT compound (Tissue-Tek; Sakura Finetek, Torrence, CA) and sectioned in a cryostat microtome (CM1900; Leica Microsystems, Concord, ON) at −20°C. Beginning 1 mm from the ora serrata and ending at the optic nerve head, transverse sections of 12-μm thickness were made with the retina being sectioned from the inner to the outer retina. The sections were placed on Superfrost Plus slides (Thermo Fisher Scientific, Ottawa, ON). The remaining eyecup was thawed and the retina was extracted. Any remaining vitreous was removed and the retina was flatmounted on a glass slide in Vectashield mounting medium (H1000; Vector Labs, Burlington, ON). 

Labelling of Retinal Ganglion Cells With BM88 Antibody

Retinal sections were washed in PBS 3 times for 10 minutes each. Then, blocking solution containing 1% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA), 1% dimethyl sulfoxide (DMSO; BDH Chemicals, Toronto, ON), and 1% Triton X-100 (BDH Chemicals) in 1 mL PBS was put on the slides for 1 hour. Between each step the slides were washed 2 times for 10 minutes each in PBS. Next, rabbit α BM88 (sc-138749 [S11], 1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:100) in blocking solution was put on the slides overnight. The primary antibody was detected with donkey α rabbit Texas Red (711-075-152, 1:100; Jackson ImmunoResearch Laboratories, West Grove, PA) in blocking solution was placed on the slides for 4 hours. The slides were coverslipped in Vectashield (H1000; Vector Labs, Burlington, ON) for visualization under an epifluorescence microscope (Zeiss Axioplan 2; Carl Zeiss, Toronto, ON). 

Measurement and Statistical Analysis of BM88 Immunoreactive RGCs

Images were captured using an AxioCamMRm camera and AxioVision 4 software (Carl Zeiss) and the number surviving RGCs were counted manually. Quantification of RGCs in flatmounts were made by counting the RGC cell bodies labeled with FG in a 92,500 mm² area randomly selected from the two remaining quadrants in the midperiphery (between 2–2.8 mm from the optic nerve head) in the hemiretina. After ONC, there was an increasing number of FG-labeled microglia and these were differentiated from RGCs based on the intensity of the staining, uniformity of the FG-labeling, ramified processes, and fusiform soma shape. 

We chose to measure staining intensity changes rather than an analysis of protein using Western blotting because BM88 was expressed throughout the retina and the changes we observed were only in one cell type in the ganglion cell layer (GCL). Such changes would not be detected using a tissue analysis. Quantitative data analysis of BM88 immunostaining in flatmounts was difficult due to the low contrast from overlapping cells in the ganglion cell layer and distal inner nuclear layer, as well as variability in the penetration of antisera through the inner limiting membrane. Cell counts collected from retinal flatmounts and sections were virtually identical (Fig. 2) after ONC. In addition, the cell counts were similar to other studies that demonstrated an initial rapid loss of RGCs followed by a protracted loss after intraorbital ONC. ^19–21 The cells were counted and the staining intensities were measured from remaining samples located in the midperiphery of the hemiretina between 1 mm from the optic nerve and 1 mm from the ora serrata. An average of RGCs from three sections was calculated per retina. The staining intensity of the antibody labeling was measured using standardized camera settings (Zeiss 100W HBO9 at 20%; gamma 1.0; 31-msec exposure; Carl Zeiss). Sections stained at the same time using the same reagents were used for intensity measurements. The exposure time of 31 msec was established by using the AxioVision (Carl Zeiss) over/under exposure function so that all the pixels were within the minimum (28 days)/maximum (0 days) linear numerical values (0–255) in all samples measured. ImageJ software (version 1.42j; National Institutes of Health [NIH], Bethesda, MD) was used to measure the average mean grey value of the whole cell on 8-bit images. The size of BM88 immunoreactive RGCs was measured using the area measurement function of Zeiss Axiovision 4 software (Carl Zeiss) on the same sections that intensity measurements were made. Cytoplasmic soma measurements were made on 230 control RGCs, 224 ONC RGCs, and 241 ONT RGCs at 2, 4, and 14 days postinjury. Statistical analysis was done using GraphPad Prism (GraphPad Software, La Jolla, CA). Significance was determined by ANOVA analysis (α = 0.05) followed by Tukey's post hoc test. All ± values were reported as 95% confidence intervals (CI). Exact P values were used to represent significance of the data, however when the exact P value was lower than 0.001, significance was represented as P < 0.001. 

Figure 2

 

Micrographs and graph showing the loss of FG containing RGCs after ONC in retinal flatmounts. At day 0 there were many RGCs containing FG (A). However, 7 (B) and 14 (C) days after ONC there were both RGCs and microglia (m) containing with FG. After 14 days following injury, RGCs were lightly labeled with FG, whereas most of the cells containing FG were transcellularly labeled microglia having short ramified processes and a fusiform soma. There was a significant decrease in the survival of RGCs 7 days after ONC in flatmounts ([D]; grey squares and dotted line). The first significant decrease in RGCs was observed 4 days after ONC. The quantification of survival RGCs after ONC was not different whether obtained from retinal flatmounts or retinal sections ([D]; black circles and solid line). Error bars: 95% CIs.

Results

RGC Loss After Optic Nerve Injury

There was an expected gradual loss of RGCs after ONC. The number of RGCs retrogradely labeled with FG decreased and the number of microglia containing FG increased 7 and 14 days after ONC (Figs. 2A–C). In retinal flatmounts, there was no significant loss of RGCs until 7 days after ONC (Fig, 2D; squares; ANOVA/Tukey's post hoc test). There were 70.29 ± 17.64% of RGCs remaining 7 days after ONC. Later in the injury, there were 36.42 ± 8.55% and 21.39 ± 12.55% RGCs remaining 14 and 28 days after ONC, respectively. There was no difference between the percentage of RGCs that were quantified using retina flatmounts (Fig. 2D; squares) or sections (Fig. 2D; circles). 

In the absence of an injury, there were 56.46 ± 3.09 RGCs/mm (n = 6; Figs. 3, 4). The RGC density 2 days after ONC was 53.06 ± 0.92 RGCs/mm, which was not significantly different from the control (n = 4; Figs. 3, 4; ANOVA/Tukey's post hoc test; P = 0.671). The RGC density 4 days after ONC was significantly lower with 47.96 ± 3.53 RGCs/mm remaining (n = 6; Figs. 3, 4; ANOVA/Tukey's; P < 0.001). At 7 days after ONC there were 30.42 ± 2.15 RGCs/mm remaining, representing a loss of 46.1% of RGCs compared with controls (n = 6; Figs 3, 4; ANOVA/Tukey's; P < 0.001). The RGC density after 14 days of ONC was 17.61 ± 1.38 RGCs/mm (n = 6; Figs. 3, 4; ANOVA/Tukey's; P < 0.001). The RGC density after 28 days of ONC was 11.15 ± 4.93 RGCs/mm, representing a loss of 80.3% of the RGCs present in the control (n = 4; Figs. 3, 4; ANOVA/Tukey's; P < 0.001). These results are similar to those shown by Parrilla-Reverter et al. ²² where there was a loss of 30% of RGCs by 7 days and 68% of RGCs by 12 days after ONC. 

Figure 3

 

Micrographs showing the downregulation of BM88 after ONC. Before injury, most labeled RGCs (gold) colocalized with BM88 (red). After injury, there was a decrease in the number of RGCs immunoreactive for BM88, which preceded the loss of RGCs.

Figure 4

 

Graph comparing the decrease in BM88 immunoreactive RGCs with the decrease in FG-labeled RGCs after ONC. In control retinas, 99% of BM88 immunoreactive RGCs were colocalized with FG. However, there were only 20% of RGCs immunoreactive for BM88 7 days after ONC. Error bars: 95% CIs.

A similar loss of RGCs occurred after the ONT injury. There was no significant difference in cell count 2 days after ONT as there were 58.22 ± 7.07 RGCs/mm (n = 4; Figs. 5, 6; ANOVA/Tukey's post hoc test; P = 0.981). However, there was a significant difference 4 days after ONT with 48.40 ± 4.69 (n = 4; Figs. 5, 6; ANOVA/Tukey's; P = 0.193). At 7 days after ONT there were 30.38 ± 1.96 RGCs/mm remaining, representing a loss of 46.2% of RGCs from the control (n = 4; Figs. 5, 6; ANOVA/Tukey's; P < 0.001). The RGC density after 14 days of ONT was 16.18 ± 3.40 RGCs/mm. The RGC density after 28 days of ONT was 7.04 ± 3.53 RGCs/mm, representing a loss of 87.53% of the cells compared with controls (n = 4; Figs. 5, 6; ANOVA/Tukey's; P < 0.001). This loss of RGCs after ONT was similar to those reported previously in the literature. ^23,24  

Figure 5

 

Micrographs showing the downregulation of BM88 after ONT. Before injury, most FG-labeled RGCs (gold) colocalized with BM88 (red). After injury, there was a decrease in the number of RGCs immunoreactive for BM88 which preceded the loss of RGCs.

Figure 6

 

Graph comparing the decrease in BM88 immunoreactive RGCs with the decrease in FG-labeled RGCs after ONT. The decrease in BM88 immunoreactive RGCs preceded the loss of RGCs following ONT. In control retinas, 99% of BM88 immunoreactive RGCs were colocalized with FG. However, there were only 48% of RGCs immunoreactive for BM88 7 days after ONT. Error bars: 95% CIs.

Colocalization of BM88 With Fluorogold

In uninjured retinas there were 55.85 ± 3.12 BM88 immunoreactive RGCs/mm, representing 98.9% of all RGCs (n = 6; Figs. 3, 4). Similarly, 2 days after ONC there were 51.39 ± 1.55 BM88 immunoreactive RGCs/mm, which was not significantly different from control (n = 4; Figs. 3, 4; t-test; P = 0.053). There was no difference 4 days after ONC as there were 43.39 ± 3.39 BM88 immunoreactive RGCs/mm (n = 6; Figs. 3, 4; t-test; P = 0.056). However, 7 days after ONC there was a significant reduction in the number of BM88 immunoreactive RGCs, with 5.96 ± 2.08 immunoreactive RGCs/mm remaining (n = 6; Figs. 3, 4; t-test; P < 0.001). This represented only 19.6 % of the RGCs that remained after 7 days of ONC. Similarly, 14 and 28 days after ONC there were only 2.54 ± 1.00 (n = 6; Figs. 3, 4; ANOVA/Tukey's post hoc test; P < 0.001) and 2.53 ± 2.32 (n = 4; Figs. 3, 4; t-test; P = 0.002) BM88 immunoreactive RGCs/mm remaining, respectively. This represented only 14.4% of the RGCs that remained after 14 days of ONC and 22.7% of the RGCs that remained after 28 days following ONC. 

A similar trend was evident for BM88 immunoreactivity after ONT, except that the downregulation began earlier. Two days after ONT there was no significant change from control with 56.22 ± 7.54 BM88 immunoreactive RGCs/mm (n = 4; Figs. 5, 6; t-test; P = 0.670). However, at 4 days there was a significant reduction with 35.56 ± 6.98 BM88 immunoreactive RGCs/mm remaining (n = 4; Figs. 5, 6; t-test; P = 0.002). This represented only 73.5% of the RGCs that remained after 4 days of ONT. The loss of BM88 immunoreactive RGCs was more pronounced 7 days after ONT where there were only 14.49 ± 3.75 BM88 immunoreactive RGCs/mm remaining (n = 4; Figs. 5, 6; t-test; P < 0.001). This means that only 47.7% of the remaining RGCs were labelled with BM88. This remained the trend at 14 and 28 days after ONT, where there were 3.94 ± 1.5 (n = 4; Figs 5, 6; t-test; P <0.001) and 1.88 ± 0.92 (n = 4; Figs. 5, 6; t-test; P = 0.005) BM88 immunoreactive RGCs/mm remaining, respectively. This represented only 24.4 % of the RGCs that remained after 14 days of ONT and 25.6 % of the RGCs that remained after 28 days following ONT. 

Staining Intensity of BM88

There were not only fewer RGCs labeled with BM88 after ONC but the intensity of the cells labeled was also diminished. ImageJ (NIH) was used to calculate the mean grey value of the BM88 immunoreactive RGCs taken at the same exposure and then expressed as a percentage staining of the control. Initially, 2 and 4 days after ONC there was a significant increase of 19.0 ± 3.6% (n = 4; Figs. 7, 8; ANOVA/Tukey's; P < 0.001) and 23.7 ± 5.4% (n = 6; Figs. 7, 8; ANOVA/Tukey's; P < 0.001), in staining intensity, respectively. This increase in staining intensity was not due to reduction in RGC size which might concentrate the protein in the cell (Fig. 11). There was no significant change in the average RGC size between controls, 4 days after ONC, and 2 days after ONT. The average RGC size in control retinas was 134.5 ± 5.3 μm² (13.1-μm diameter; n = 3; ANOVA/Tukey's), which is agreement with the average RGC size reported by Danias et al. ²⁵ (13.7-μm diameter vs. 13.1 μm) and Urcola et al. ²⁶ (134.3-μm² area vs. 134.5 μm²). The size did not change 4 days after ONC where the size was 133.8 ± 6.4 μm² (n = 3; ANOVA/Tukey's) and 2 days after ONT where the size was 139.0 ± 5.0 μm² (n = 3; ANOVA/Tukey's). However, 7 days after ONC there was a sharp decline in staining intensity to 55.28 ± 5.24% of the control (n = 6; Figs. 7, 8; ANOVA/Tukey's; P < 0.001). This decrease persisted for 28 days after ONC, where the staining intensity was 41.07 ± 3.76% of the control after 14 days of ONC and 38.43 ± 3.95% (n = 6; Figs. 7, 8; ANOVA/Tukey's; P < 0.001) of the control after 28 days of ONC (n = 4; Figs. 7, 8; ANOVA/Tukey's; P < 0.001). At 14 days, following ONC or ONT there was a significant decrease in RGC size (Fig. 11). A 37.7% decrease in RGC area was observed 14 days after ONC (83.8 ± 6.7 μm²; n = 3; ANOVA/Tukey's; P < 0.001) and a 47.2% decrease in RGC area observed 14 days after ONT (71.1 ± 9.6 μm²; n = 3; ANOVA/Tukey's; P < 0.001). 

Figure 7

 

Micrographs showing the downregulation of BM88 after ONC. Before injury, most RGCs colocalized with BM88. Early after the injury (at 4 days), there was an increase in the staining intensity of BM88. After 7 days of injury, there was a decrease in the number of RGCs immunoreactive for BM88 and a decrease in the staining intensity of BM88 before the loss of the RGCs.

Figure 8

 

Graph showing the decrease in staining intensity of BM88 after ONC. Four days after injury there was an increase of 24% in the staining intensity of BM88. However, after 7 days of injury, there was a 55% decrease in the staining intensity of BM88. From 14 days of injury the staining intensity dropped to its lowest point and this remained constant over the 28-day period. Error bars: 95% CIs.

A similar change in BM88 staining intensity was observed after ONT, except that the decrease in intensity occurred after 4 days following ONT instead of 7 days, as seen after ONC. Early in the injury, there was a significant increase of 7.9 ± 2.7% in staining intensity after 2 days following ONT (n = 4; Figs. 9, 10; ANOVA/Tukey's; P < 0.001). Then, beginning 4 days after ONT, there was a decline in staining intensity where it was 54.74 ± 2.51% of the control (n = 4; Figs. 9, 10; ANOVA/Tukey's; P < 0.001). This decrease persisted where the staining intensity was 49.33 ± 1.99% of the control 7 days after ONT, 55.34 ± 5.29% of the control 14 days after ONT, and 51.04 ± 11.65% of the control 28 days after ONT (n = 4; Figs. 9, 10; ANOVA/Tukey's; P < 0.001). 

Figure 9

 

Micrographs showing the downregulation of BM88 after ONT. Before injury, most RGCs colocalized with BM88. Two days after injury there was an increase in the staining intensity of BM88. This was earlier than seen after ONC. After 4 days of injury, there was a decrease in the number of RGCs immunoreactive for BM88 and a decrease in the staining intensity of BM88 before the loss of RGCs.

Figure 10

 

Graph showing the decrease in staining intensity of BM88 after ONT. Two days after injury there was an increase of 8% in the staining intensity of BM88. However, after 4 days of injury, there was a 55% decrease in the staining intensity of BM88 and this remained constant over the 28-day period. Error bars: 95% CIs.

Figure 11

 

Retinal ganglion cell size was measured at time points when an increase in staining intensity of BM88 was observed (4 days after ONC and 2 days after ONT) and later after injury, when a decrease in staining intensity was observed (14 days after ONC and ONT). There was no significant difference in RGC size 4 days after ONC or 2 days after ONT, suggesting that the observed increase in BM88 staining intensity early in injury was not due to cell shrinkage, resulting in a concentration of the protein. However, 14 days after either ONC or ONT, when a decrease in BM88 staining intensity was observed, there was a significant decrease in cell size (*). ANOVA/Tukey's post hoc tests; *P < 0.001; Error bars: 95% CIs.

This decrease in staining intensity of BM88 after optic nerve injury was only seen in RGCs. Horizontal cells are another cell type in the retina that also express BM88 (Fig. 1). There was no change in BM88 staining intensity in horizontal cells after 4 to 28 days after ONC (Figs. 12, 13; ANOVA/Tukey's). Four days after ONC the staining intensity was 101.80 ± 4.65% of the control. Similarly, the staining intensity of BM88 in horizontal cells 7, 14, and 28 days after ONC was 92.70 ± 2.50%, 90.74 ± 2.09%, 93.84 ± 8.46% of the control, respectively. 

Figure 12

 

Micrographs showing BM88 immunolabeling in the outer retina after ONC. Unlike the intensity changes observed in the inner retina, there was no change in immunoreactivity associated with punctate labeling in the OPL (arrowhead) or HC somas (arrow) at 4, 7, and 14 days after ONC. All micrographs are at the same magnification.

Figure 13

 

Graph showing staining intensity of BM88 in horizontal cells after ONC. There was no significant difference in BM88 staining intensity in the OPL between the control and 4 to 28 days after ONC.

Discussion

The GCL of the retina contains both RGCs and displaced amacrine cells in approximately equal proportions. ^27,28 This makes it difficult to perform quantitative analyses of RGC loss, because a measure of total cell number will underestimate RGC loss due to the large number of uninjured displaced amacrine cells. The standard for labeling RGCs has been to use retrogradely transported tracers injected into the superior colliculus, such as FG. ^17,29 The use of antibodies has been attractive because it is less labor intensive and reduces the amount of invasive procedures performed on the animals. One such antibody that has been used for this purpose is Thy1.1. The problem with using Thy1.1 as a marker of RGC is that its expression pattern changes after injury. ^15,16 Initially BM88 appeared to be a good marker for uninjured RGCs because it labeled nearly all FG-labeled cells and no displaced amacrine cells. However, 4 to 7 days after optic nerve injury, the number of BM88 immunoreactive RGCs started to decrease, prior to the decrease in the number of FG labeled RGCs. The staining intensity decreased earlier after ONT than ONC (4 vs. 7 days) likely because an ONT is a more serve injury ²¹ and, therefore, intracellular changes may occur sooner. The early change in BM88 staining intensity could not be accounted for by a decrease in cytoplasmic volume that might concentrate protein. We observed a decrease in BM88 immunoreactive RGC size after 14 days in both ONC and ONT lesioned retinas. However, there was no significant change in RGC size 4 days after ONC or 2 days after ONT, times when increases in BM88 staining intensity were observed. Although there are a few exceptions, an early decrease in cell volume (apoptotic volume decrease [AVD]) is a hallmark of cells undergoing apoptosis such as RGCs that have been injured by ONC or ONT. ³⁰ The timing of the early and late AVD depends on the cell type and injury. Janssen et al. ³¹ observed a decrease in nuclear size that correlated with soma size as early as 1 day after optic nerve crush in the mouse and a 25% decrease in nuclear and soma size between 3 and 5 days. They also observed a decrease in nuclear size among nonganglion cells that do not undergo apoptosis. Our observation of a 38% decrease in RGC size 14 days after injury is consistent with the earliest reports of RGC shrinkage after ONC in the rat (50% at 11 days ³² ; 34% at 14 days ^19,33 ). We did not detect a decrease in average RGC size early after injury. In contrast, Moore and Thanos ³⁴ observed a 34% and 56% increase in RGC size 14 days after open or blind ONC in the rat. A transient decrease in RGC size was followed by a 16% increase in size at 14 days and an 8% increase at 28 days following ONC in the cat. ³⁵ In primate glaucoma, ³⁶ human glaucoma, ³⁷ and in the DBA/2J mouse model of glaucoma ³⁸ there were modest or no changes in RGC size over the time course studied. It has been suggested that a protracted early phase of AVD may explain why the proportion of large RGCs appears to decrease, while the proportion of medium to small RGCs remains stable after injury. ³⁹ However, it has been argued that RGC apoptosis is a rapid event ³⁸ occurring in less than 1 hour in rat retinal explants. ⁴⁰ Using detection of apoptosing retinal cells (DARC) imaging, Guo and Cordiero ⁴¹ have shown that fewer than 10% of RGCs undergo apoptosis between 0 and 7 days following ONT in the rat. This may explain why we did not detect a significant decrease in RGC size at 2 or 4 days following ONT or ONC. The loss of Brn3a immunoreactive RGCs has also been shown to begin earlier after ONT than ONC. ⁴² At 7 days after optic nerve injury there were only 20% to 50% of RGCs still immunoreactive for BM88 and of these cells the expression of BM88 (as measured by staining intensity) was 49% to 55% of the control. This decrease persisted 14 and 28 days after optic nerve injury. Therefore, BM88 is not a good marker for the purposes of quantifying remaining RGCs after injury, however, examination of changes in its expression may be a useful marker of abnormal RGC function that precedes RGC loss. Brn3 (Pou4f family of transcription factors) have also been investigated as a marker of RGCs. ^42,43 The Brn3a subtype has been shown to be a good marker of RGCs in control and injured retinas, however, Brn3b and Brn3c expression decreased below the intensity required for quantification. ^42,43 Brn3a colocalized with most FG containing RGCs, however, Western blot analysis showed that Brn3a expression was downregulated after injury even though the staining is detectable in the retina. ⁴² This downregulation in Western blotting was attributed to a dilution effect since RGCs only represent less than 1% of the retina cell population. ⁴² We have shown that the loss of BM88 immunoreactive RGCs precedes that of Brn3a immunoreactive RGCs in an injured retina, indicating that BM88 expression is downregulation before the loss of RGCs. 

We have shown that after optic nerve injury there was a reduction in the number of RGCs immunoreactive for BM88 and that there was a reduction in the staining intensity of RGCs that still express BM88. However, early in the injury there was an increase in BM88 staining intensity. Four days after ONC and 2 days after ONT there was an increase of 20% and 7.9%, respectively, in staining intensity compared with controls. This suggests that the early changes in the physiological health of the RGCs before the death of the cell can be revealed by BM88 expression. This initial increase may be an abortive attempt by the cell to survive after the injury. An increase in BM88 expression may help reduce calcium mobilization by increasing calcium uptake by the mitochondria and lowering the calcium storage in the endoplasmic reticulum. ⁵ Experiments using BM88 overexpressing neuroblastoma cells show that BM88 may have a neuroprotective role against neurotoxic stimuli through the inhibition of ERK and BAX. ³  

The downregulation of such an important suppressor of entry into the cell cycle raises many questions. It could suggest that RGCs, after axonal injuries, may attempt to re-enter the cell cycle, which leads to cell death. Cell cycle molecules have been shown to be involved in normal developmentally-associated neuronal death. ^44,45 In normal development, approximately 50% of neurons undergo cell death. ⁴⁶ Neuronal survival also heavily relies on competition for target-derived trophic factors. ^47–50 After loss of the synaptic target of postmitotic cerebellar granule cells, this resulted in cell death that was associated with elevation of cyclin D and other cell-cycle proteins. ⁵¹ It seems as if first gap phase/synthesis gap phase (G1/S)-related events are required for neuronal death during development. When the transition to this phase was blocked, it rescued neurons from cell death caused by nerve growth factor (NGF) deprivation. ^52,53 Deprivation of NGF in sympathetic neuron cultures has been shown to be associated with an increase in transcripts encoding cyclin D1. ⁵⁴ BM88 is an important inhibitor of cyclin D1 and may therefore play a role in regulating apoptosis of neurons. Neurotrophic deprivation is also thought to play an important role in glaucoma ⁵⁵ and, therefore, apoptosis after neurotrophic deprivation may be regulated by cell-cycle molecules, such as BM88 and cyclin D1. There are examples of neurons re-entering the cell cycle in other neurodegenerative disorders, such as Alzheimer's disease. ⁵⁶ This type of dysregulation of the cell cycle is known to put neurons into an apoptosis prone state. ³ Cell cycle–related proteins are found to be elevated in neurons that are at risk to die in many different neurodegenerative disorders. ^57,58 Many cell cycle–associated genes and proteins have shown to be upregulated after optic nerve injury, including RAD9, PPM1B, Cyclin D, Cyclin G1, aryl hydrocarbon, and Rb proteins. ^59,60 In addition, cyclin D1 is expressed in high levels in lesioned hippocampal neurons suggesting that BM88 acts as a modulator of apoptosis. ⁶¹ All this taken together implicates BM88 as an important suppressor of the cell cycle in mature neurons and its dysregulation may cause cells to be more prone to apoptosis. Future studies should examine the interaction between BM88 downregulation and other proteins involved in the cell cycle such as cyclin D1 and the effects of maintaining BM88 expression on RGC survival. 

BM88 is also thought to play roles in calcium buffering and helping to maintain the integrity of the mitochondria. ⁵ Calcium regulation in neurons is important because calcium overload in the cytoplasm and mitochondria leads to apoptosis. ⁶² BM88 acts to block calcium release from calcium stores in the cell by modulating IP₃ and IP₃R. ⁵ Downregulation of BM88 in RGCs may cause the release of calcium from intracellular stores leading to apoptosis of the cell. Blocking voltage-dependent calcium channels with Flunarizine has been shown to enhance RGC survival after axotomy in rats. ⁶³ BM88 is also found on the membrane of mitochondria but its role there is not well understood. ¹ It is has been suggested that BM88 inhibits ERK, leading to BAX not being able to bind to the mitochondria. ⁵ When BAX binds to the mitochondria, this initiates the pathway for apoptosis. ⁶⁴ There are therefore three mechanisms by which downregulation of BM88 may lead to RGC cell death, impaired calcium regulation, initiation of apoptosis and mitochondrial dysfunction, and cell-cycle dysregulation. 

This study has shown that BM88, like many other proteins, is downregulated after optic nerve injury. Recently, isobaric tags for relative and absolute quantification (iTRAQ) analysis showed that 110 of 337 proteins that were identified were differentially regulated after optic nerve injury (58 upregulated and 46 downregulated). ⁶⁵ It is important to study the different proteins that are differentially regulated at different times in the injury because these proteins can have neuroprotective effects after injury. For example, Thymosin-β4 was upregulated early after injury and its administration can increase RGC survival and enhance axonal regeneration. ⁶⁵ Therefore, further investigation into the role of BM88 in RGC survival will aid in the development of therapeutics that can be beneficial in optic neuropathies such as glaucoma. 

Acknowledgments

The authors thank Laurie C. Doering, PhD, for access to the Neuroscience Imaging Facility. 

Supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant 171190-2008, Glaucoma Research Society of Canada, and Canadian Health Institute of Health Research Scholarship (TFS). 

Disclosure: A.M. Siddiqui, None; T.F. Sabljic, None; P.D. Koeberle, None; A.K. Ball, None