Hyperthermic pre-conditioning protects retinal neurons from N-methyl-d -aspartate (NMDA)-induced apoptosis in rat

Brain Research 970 (2003) 119–130 www.elsevier.com / locate / brainres

Research report

Hyperthermic pre-conditioning protects retinal neurons from N-methyl-D-aspartate (NMDA)-induced apoptosis in rat Jacky M.K. Kwong a,b , *, Tim T. Lam b,c , Joseph Caprioli a a

Department of Ophthalmology, Jules Stein Eye Institute, University of California Los Angeles School of Medicine, Room B-121, 100 Stein Plaza, Los Angeles, CA 90095 -7000, USA b Department of Ophthalmology and Visual Sciences, the Chinese University of Hong Kong, Hong Kong, Hong Kong c Department of Ophthalmology, Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Accepted 14 January 2003

Abstract Glutamate-induced excitotoxicity is associated with a selective loss of retinal neurons after retinal ischemia and possibly in glaucoma. Since heat shock protein (HSP) 70 is known to play a protective role against ischemic neuronal injury, which is also linked to excitotoxicity, we studied the expression of inducible (HSP72) and constitutive (HSC70) forms of HSP70 in apoptosis of retinal ganglion cells (RGCs) after intravitreal injection of 8 nmoles N-methyl-D-aspartate (NMDA), a glutamate receptor agonist. Approximately 18 h after NMDA injection, there were increased numbers of TUNEL-positive cells and cells with elevated HSP72 immunoreactivity in the retinal ganglion cell layer (RGCL), but there were no noticeable changes in HSC70 immunoreactivity. These HSPs positive cells were also Thy-1 positive, a marker for RGCs. Hyperthermic pre-conditioning, which is known to induce HSPs, given 6 or 12 h prior to NMDA injection ameliorated neuronal loss in the RGCL as counted 7 days after NMDA injection but pre-conditioning at 18 h prior to NMDA injection did not have any ameliorative effect. Quercetin, an inhibitor of HSP synthesis, abolished the ameliorative effect of hyperthermic pre-conditioning. Pre-conditioning elevated HSP72 but not HSC70 immunoreactivity and reduced the number of TUNEL-positive cells in the RGCL at 18 h. Our results suggest that intravitreal injection of NMDA induces an up-regulation of HSP72 in a time-dependent manner but not HSC70 in RGCs, indicating a stress response of HSP72 in RGCs and other inner retinal neurons after exposure to NMDA. Hyperthermic pre-conditioning given within a therapeutic window is neuroprotective to the retina against NMDA-induced excitotoxicity, likely by inhibiting apoptosis through the modulation of HSP72 expression.  2003 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: others Keywords: Heat shock protein; Ganglion cell; Glaucoma; Apoptosis; Excitotoxicity; N-Methyl-D-aspartate

1. Introduction The stress response includes up-regulation of heat shock proteins (HSPs) and is highly conserved in all organisms from bacteria to mammals. In the central nervous system (CNS), gene expression of HSPs can be induced by physiological and environmental challenges [6,35]. These protein families maintain cellular integrity and viability,

*Corresponding author. Tel.: 11-310-206-7900; fax: 11-310-2067773. E-mail address: [email protected] (J.M.K. Kwong).

and protect neurons from a wide range of harmful conditions [52]. Among the various HSP families classified according to their molecular weights, the HSP70 family has been shown to protect neurons from ischemic insults and seizures [11,25]. The constitutive form of the HSP70 family is HSC70, which functions as a molecular chaperone and maintains normal protein folding to prevent their damage, promotes intracellular processing of newly synthesized proteins, facilitates protein translocation, and enhances protein degradation [10,53]. On the other hand, HSP72, the inducible form of HSP70, prevents protein aggregation and acts as a protein chaperone in response to various noxious

0006-8993 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02298-4

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stimuli, such as heat stress, activated oxygen and nitrogen intermediates, heavy metal exposure, inflammatory mediators or infection [36,44,17,16]. Neurons in transgenic mice that over-express HSP72 [45,28] or neurons in rats injected with the herpes virus containing the HSP72 gene [65] are more resistant to oxygen-glucose deprivation, glutamate toxicity, ischemia and seizures. The survivalpromoting effect of the HSP70 family of proteins has been linked to their ability to inhibit apoptosis, the highly regulated cell death process involved in stroke, ischemia and many neurodegenerative diseases [52,32]. However, the exact interactions between HSP70 proteins and the apoptotic pathways are not well understood [3,18]. Excitotoxicity has been linked to stroke, cerebral ischemia, hypoglycemia, trauma, epilepsy, and neurodegenerative diseases such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and acquired immunodeficiency syndrome [30]. Excitotoxicity in retinal ganglion cells (RGCs) and other inner retinal neurons has been examined and shown to be caused predominantly by overstimulation of the N-methyl-D-aspartate (NMDA) receptor, a subtype of the glutamate receptor [57,58,21], involving apoptosis [26]. Earlier studies by Dreyer et al. [14] demonstrated elevated levels of glutamate in the vitreous bodies of patients with glaucoma and monkeys with experimental glaucoma suggesting a possible role of glutamate induced retinal excitotoxicity in glaucoma. However, the validity of Dreyer and co-workers’s data was questioned recently [12] and a more recent study by Carter-Dawson et al. [9] noted no elevation of vitreal glutamate level in monkeys with experimental glaucoma. Hence, whether there is an elevated intravitreal level of glutamate in glaucoma remains unclear. Nonetheless, excitotoxicity of inner retinal neurons remains a viable explanation for secondary axonal injury and cell death in glaucoma [19,48,63], retinal ischemia–reperfusion injury [42] and other optic neuropathies [51]. To examine a possible endogenous neuroprotective role of HSPs in inner retinal neurons, especially the RGCs, we studied the effects of NMDA injection on HSP expression in RGCs and hyperthermic pre-conditioning, a means to induce HSP synthesis, with and without quercetin, an inhibitor of HSP synthesis, on RGC survival after intravitreal injection of NMDA using cell counting in the retinal ganglion cell layer (RGCL), TdT-mediated biotin-dUTP nick end labeling (TUNEL) and immunohistochemical analysis of HSP72 and HSC70. Laser confocal scanning microscopy was used to study double-labeling of TUNEL and HSP72 or HSC70.

2. Material and methods

2.1. Experimental design Forty-five- to 50-day-old adult male Sprague–Dawley

rats, each weighing 200–250 g, were used in this study. All experiments were approved by the Animal Research Committee of the University of California, Los Angeles and performed in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. To examine the involvement of HSP72 and HSC70 in NMDA-induced retinal excitotoxicity, animals were injected with 2 ml of 4 mM NMDA intravitreally and then euthanized with an overdose of pentobarbital at 4, 8, 12, 18, 24, 36, or 48 h after injection. The eyes were enucleated and processed for TUNEL assay and immunohistochemical analysis. Quantitative analyses of TUNEL and immunostaining of HSP72 and HSC70 were performed (see below). Immunofluorescent stained sections with antibodies against HSP72 or HSC70 and TUNEL were examined with laser confocal scanning microscopy. To examine the effect of hyperthermic pre-conditioning in NMDA-induced excitotoxicity, animals were divided into eight groups. Four groups of animals were subjected to hyperthermia 0, 6, 12 or 18 h prior to intravitreal injection of NMDA. One group was given systemic administration of quercetin and hyperthermia 12 h prior to NMDA injection. The remaining three groups served as controls: normal untreated rats, hyperthermia-treated rats that were intravitreally injected with phosphate-buffered saline (PBS) in place of NMDA, and non-hyperthermiatreated, NMDA-injected rats. To examine cell survival in the RGCL, animals from each group were euthanized 7 days after injection of NMDA or PBS, and the enucleated eyes were collected for flat preparations of retinas and cell counting. To explore the role of HSP72 and HSC70 in hyperthermic pre-conditioning, animals from each of the four experimental groups (described above) were euthanized 18 h after NMDA injection. To evaluate the effect of hyperthermic pre-conditioning, additional groups of animals were given hyperthermia 6, 12 or 18 h prior to intravitreal injection of PBS and euthanized 18 h after PBS injection. The enucleated eyes were subjected to TUNEL assay and immunohistochemical analysis with antibodies against HSC70 or HSP72 as described below.

2.2. Administration of NMDA The procedure published by Siliprandi et al. [54] was modified to induce excitotoxic cell death in the inner retinas of rats [26]. Briefly, anesthetized animals were given topical 0.5% proparacaine hydrochloride and intravitreally injected with 2 ml of 4 mM, corresponding to 8 nmoles, neutralized NMDA (Sigma, St. Louis, MO) in sterile PBS (0.1 M) [26]. The same volume of PBS was given to a group of control animals. Topical tobramycin 0.3% ointment (Tobrex, Alcon Laboratories Inc., Fort Worth, TX) was applied prophylactically.

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2.3. Immunohistochemical and quantitative analysis The enucleated eyes were bisected vertically and fixed overnight in Davidson’s fixative (32:2.2:11:54.8, ethanol:neutral buffered formalin:glacial acetic acid:distilled water, by volume) [22,27]. They were then processed and embedded in paraffin. Then, 4 mm-thick sagittal retinal sections were prepared for TUNEL assay and immunohistochemical analysis. Only those retinal sections along the vertical meridian of the optic nerve head were collected for the study. Adjacent retinal sections were used for immunohistochemical analysis with a mouse monoclonal antibody against HSP72 (inducible form, StressGen Biotechnologies Corp., Victoria, BC, Canada) (1:500) and a Vectastain ABC Kit (Vector Laboratories, Inc., Burlingame, CA) as previously described [43]. Diaminobenzidine was used for color development. A negative control was included by incubating sections with blocking solution without the primary antibody. Retinal specimens collected 24 h after induction of hyperthermia were used as positive controls. A rat monoclonal antibody against HSC70 (StressGen Biotechnologies Corp.) (1:200) was used to detect the constitutive form of the HSP70 family. Immunoreactivity of HSP72 and HSC70 in the RGCL was analyzed in two ways. First, HSP72-positive or HSC70-positive cells in the RGCL of each whole length retinal section were counted manually with a light microscope. The numbers obtained by two examiners working in a masked fashion were averaged for each section. The numbers obtained from two retinal sections of each eye were then averaged and expressed as the number of positive cells per section. Second, the relative intensity of positively labeled cells of the RGCL was measured with a computer-assisted image processing unit (Image-Pro Plus software, Media Cybernetics, Silver Spring, MD) using the ‘count-measure’ function. Briefly, a digital camera (Coolsnap, RS Photometrics, Tucson, AZ) attached to the microscope (Axioplan, Carl Zeiss, Oberkochen, Germany) captured images of the immunostained sections at 6303 magnification under oil immersion. The system was calibrated according to the supplier’s manual before the analysis. For each digital image, all individual cells in the RGCL were marked by an examiner and the optical density of each cell was assessed by the computer. The intensities of approximately 60 to 80 cells per retinal section were measured and averaged to yield a single value representing one retina.

2.4. In situ TUNEL assay and morphometry of TUNELpositive cells For each rat eye, the TUNEL assay was performed on the first and the fourth serial retinal sections with the ApopTag Peroxidase In Situ Apoptosis Detection kit (Intergen Company, Purchase, NY). Diaminobenzidine

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was used as the color substrate. TUNEL-positive cells in the RGCL of each retinal section were counted with light microscopy. The numbers obtained by two examiners working in a masked fashion were averaged for each section. The numbers obtained from the two retinal sections of each eye were averaged and expressed as the number of TUNEL-positive cells per section.

2.5. Double labeling and laser confocal scanning microscopy Fluorescence immunohistochemistry was performed with anti-biotin Cy-3 conjugate or FITC conjugate (both from Sigma). After immunofluorescent staining of HSP72 or HSC70, the same sections were stained with a goat polyclonal antibody against Thy-1 (Research Diagnostics, Inc., Flanders, NJ) (1:400), a marker of RGCs. Similarly, TUNEL was performed on sections already immunolabeled with HSP72 or HSC70 antibody to label degenerating cells with ApopTag Fluorescein In Situ Apoptosis Detection kit or ApopTag Red In Situ Apoptosis Detection kit (Intergen). Immunofluorescence was examined with a Leica TCS SP laser confocal scanning microscope, a Leica DM-IRBE inverted microscope stand (Leica, Heidelberg, Germany), an argon laser for excitation at 488 nm, and a krypton laser for excitation at 568 nm. Digital images from at least three different retinal specimens per experimental group were recorded and analyzed. Two-dimensional projections of a series of five consecutive images from retinas of the experimental groups were compared to normal control retinas to study fluorescent immunoreactivity.

2.6. Hyperthermic pre-conditioning ( heat shock) Previous studies have demonstrated that hyperthermia robustly induces HSP72 in cells of the RGCL [61] and in other retinal layers in the rat [61,2]. A published procedure [43] for hyperthermic pre-conditioning was modified for the present study. In brief, an intraperitoneal injection of 400 mg / kg chloral hydrate was given to each animal. The anesthetized animals were placed in a hollow, buoyant aluminum receptacle (183837 cm) in a water bath kept at 42 8C. Body temperature was monitored with a rectal thermometer, allowed to rise to 41 8C and maintained between 41 and 42 8C for 15 min. The rats were then removed from the receptacle and allowed to recover on a warm plate at 37 8C. The awakened rats were kept at room temperature. To inhibit HSP synthesis, quercetin (4 mg / kg; Sigma, St. Louis, MO), a HSP inhibitor, was given intraperitoneally [43].

2.7. Flat preparation of retinas and cell counting in RGCL Previously described procedures [26] were used for flat

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preparations of retinas, cresyl violet staining, and cell counting. Morphologically distinguishable glial cells and vascular endothelial cells were not counted. The cell count was performed with the eyepiece reticule at 4003 magnification. Each quadrant (superior, temporal, inferior and nasal) of each retina was sampled at three regions: 1 mm (central), 2 mm (mid-periphery) and 3 mm (periphery) from the center of the optic nerve head. Three microscopic fields (0.2330.23 mm 2 ) were sampled from each of the 12 resulting regions, for a total of 36 microscopic fields, representing approximately 3.0–3.8% of the total retinal area. The counted cells were referred to as RGC-like neurons and the counting was expressed as the number of cells per mm 2 .

2.8. Statistical analysis All data are expressed as the mean6S.E.M. One-way ANOVA was performed, followed by Student’s t-test. P, 0.05 was considered significant.

3. Results

3.1. Expression of HSP72 after intravitreal injection of NMDA In the normal retina, mild HSP72 immunoreactivity (Fig. 1A) and strong HSC70 immunoreactivity (Fig. 1C) were noted in many cells of the RGCL. Eighteen hours after a single intravitreal injection of NMDA, intense HSP72 immunoreactivity was noted in the cytoplasm of many cells of the RGCL (Fig. 1B). In contrast, there was no apparent difference in HSC70 immunoreactivity in retinas after NMDA injection (Fig. 1D) compared with the normal control (Fig. 1C). No noticeable change of HSP72 or HSC70 was detected in other retinal layers (data not shown; at least five retinal sections per group were examined). To quantify these changes, the number and the optical density of immunopositive cells were counted manually and measured with a computer assisted image processing system respectively. After an injection of

Fig. 1. Immunohistochemistry of HSP72 and HSC70 in the RGCL of rat retinas 18 h after NMDA injection. Mild HSP72 immunoreactivity (arrowhead) was shown in normal control retina (A). There was increased HSP72 immunoreactivity (arrows) after NMDA injection (B). Consistently intense HSC70 immunoreactivity (arrowhead) was noted in normal control (C) and NMDA-injected (D) retinas. The central retinas were shown as representatives. RGCL, retinal ganglion cell layer; IPL, inner plexiform layer.

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NMDA, the number (Fig. 2A) and the optical density (Fig. 2C) of HSP72 immunopositive cells in the RGCL showed time-dependent changes exhibiting significantly higher numbers at 18–24 h and higher optical density at 12–18 h, respectively. In contrast, despite the moderate-to-strong HSC70 immunoreactivity seen in normal control retinas and NMDA-injected retinas, there was no significant change in the number (Fig. 2B) or the optical density (Fig. 2D) of HSC70 immunopositive cells at any time after NMDA injection. No observable changes in HSP72 or HSC70 immunoreactivity were noted in other retinal cells after intravitreal injection of NMDA (data not shown). Fig. 3 shows immuno-colocalization of HSP72 or HSC70 and Thy-1, a marker for RGCs. In a section obtained at 18 h after intravitreal NMDA injection, a time when maximal apoptotic changes occurred [26], HSP72 immunoreactivity colocalized with Thy-1-positive cells in the RGCL, indicating that the RGCs expressed increased HSP72 (Fig. 3A–C). Similarly, colocalization of HSC70 and Thy-1 was also observed in most cells in the RGCL of normal control retinas (Fig. 3D–F), suggesting that HSC70 was constitutively present in the RGCs of the normal retinas.

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3.2. In situ TUNEL labeling after intravitreal injection of NMDA Paralleling the temporal changes in HSP72 immunoreactivity and consistently our previous findings [26], the number of TUNEL-positive cells in the RGCL also showed a time-dependent change between 8 and 48 h after NMDA injection with significantly increased numbers at 12, 18 and 24 h (Fig. 4; P,0.05) exhibiting a maximum at 18 h; no TUNEL-positive cells were found in normal control retinas. To explore the relationship of HSP72 and cell death, double labeling of HSP72 and TUNEL was performed in the same retinal sections. Analysis of HSP72 and TUNEL colocalization in the RGCL 18 h after NMDA injection revealed three patterns: (1) some TUNEL-negative cells showed moderately increased HSP72 immunoreactivity (arrows; Fig. 5A–C); (2) some TUNEL-positive cells showed intensely increased HSP72 immunoreactivity (arrow; Fig. 5D–F); and (3) the remaining TUNEL-positive cells were weakly labeled (arrowheads; Fig. 5A–C) or unlabeled (arrowhead; Fig. 5D–F) by HSP72 antibody. In contrast, at the same time point after NMDA injection (Fig. 5G–I), both TUNEL-positive (arrow) and TUNEL-

Fig. 2. Quantification of immunoreactivity of HSP72 (A and C) and HSC70 (B and D) in the RGCL of retinas after NMDA injection. (A) Counting of HSP72 positive cells. There was a gradual increase in the number of HSP72 positive cells after NMDA injection, showing statistically significant increases at 18 and 24 h (* P,0.05) and a return to normal at 36 and 48 h. (B) Counting of HSC70 positive cells. There were no significant changes throughout the observation period (P50.46). (C) Intensity of HSP72 labeling. Note increased optical density of HSP72 immunoreactivity 12 and 18 h after NMDA injection (* P,0.05). (D) Intensity of HSC70 labeling. There was no remarkable change of HSC70 (P50.53). Values represent mean6S.E.M. of measurements in eight retinas.

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Fig. 3. Colocalization of HSP72 or HSC70 and Thy-1. Immunolabeling of HSP72 (A, C; red) and Thy-1 (B, C; green) in the retina 18 h after NMDA injection (A–C) and of HSC70 (D, F; red) and Thy-1 (E, F; green) in normal control retina (D–F). Eighteen hours after NMDA injection, the increased HSP72 immunoreactivity (A) and positive labeling of Thy-1 (B) were co-localized (arrows; C) in the RGCL. Note positive labeling of HSC70 (D), Thy-1 (E), and their co-localization (arrows; F) in the RGCL of normal control retina. Each image is a two-dimensional projection of a series of five consecutive images with a single-image plane equal to 1 mm in depth.

Fig. 4. Morphometry of TUNEL-positive nuclei in the RGCL of retinas after NMDA injection. Significant increases in the number of TUNEL-positive nuclei were noted from 12 and 24 h after injection compared to normal control. Values represent mean6S.E.M. of measurements in eight retinas (* P,0.05).

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Fig. 5. Colocalization of HSP72 or HSC70 and TUNEL. Immunolabeling of HSP72 (A, C; green) (D, F; red), HSC70 (G, I; green), and TUNEL (B, C, H, I; red) (E, F; green) in the RGCL of the retinas 18 h after NMDA injection. Immunostaining of HSP72 and TUNEL showed cells with increased HSP72 immunoreactivity and negative TUNEL (arrows) and TUNEL-positive cells with mild HSP72 immunoreactivity (arrowheads) (A–C). Immunostaining of HSP72 and TUNEL showed a TUNEL-positive cell with intense HSP72 immunoreactivity (arrow) and a TUNEL-positive cell in the absence of HSP72 immunoreactivity (arrowhead) (D–F). Immunostaining of HSC70 and TUNEL showed a cell with co-localization of HSC70 and TUNEL (arrow) and an HSC70-positive cell without TUNEL (arrowhead) (G–I). Each image is a two-dimensional projection of a series of five consecutive images with a single-image plane equal to 1 mm in depth.

negative cells (arrowhead) in the RGCL showed HSC70 immunoreactivity.

3.3. Effects of hyperthermic pre-conditioning in NMDAinduced excitotoxicity To explore a possible neuroprotective effect of HSP72, we performed hyperthermic pre-conditioning to induce HSP72 expression at various times prior to NMDA injection. At 18 h after NMDA injection, when the number of TUNEL-positive cells, an index of apoptotic cell death, was at a maximum according to our previously published results [26], there were fewer TUNEL-positive cells in the RGCL of retinas that had been pre-conditioned with hyperthermia 6 or 12 h before NMDA injection (Fig. 6B) (less apoptosis) than in the RGCL of those NMDA-injected retinas without hyperthermic pre-conditioning (Fig. 6A).

Table 1 shows the results of counting these TUNELpositive cells under various conditions. There was a significant reduction (P,0.05) in the number of TUNELpositive cells in the RGCL of retinas subjected to hyperthermic pre-conditioning 6 or 12 h (neuroprotective effect) but not 18 h before NMDA injection (Table 1). Simultaneous administration of quercetin and pre-conditioning at 12 h prior to NMDA injection abolished the reduction of TUNEL-positive cells in RGCL. Very few TUNEL-positive cells were present in the RGCL of hyperthermia pre-conditioned control retinas (with PBS injections or no NMDA injection as shown in Fig. 6C) or normal control retinas. At the same time point, when compared with the normal control retinas, there were significant differences in the number (Table 1) and optical density (data not shown) of HSP72-positive cells of all experimental retinas (retinas

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Fig. 6. TUNEL and flat preparations of retinas. (A–C) Composite TUNEL micrograph of retinal sections obtained at 18 h after NMDA injection without hyperthermia (A), with hyperthermia induced 12 h before NMDA injection (B), or with hyperthermia and PBS injection (C). Fewer TUNEL-positive cells (arrow) were noted in the RGCL of the pre-conditioned retina after NMDA injection. (D–F) Composite micrograph of representative flat preparations of retinas 7 days after NMDA injection stained by cresyl violet without hyperthermia (D), with hyperthermia prior to NMDA injection (E), and with hyperthermia but with PBS injection in place of NMDA (F). There were more RGC-like neurons (arrows) in the retina subjected to hyperthermic pre-conditioning (E; 12 h before NMDA injection) than in the non-pre-conditioned retina (D). Similar numbers were noted in both NMDA- and PBS-injected retina after hyperthermia. Central retinas were shown as representatives. RGCL, retinal ganglion cell layer; IPL, inner plexiform layer.

treated with NMDA; retinas treated with NMDA plus hyperthermic pre-conditioning; or retinas treated with hyperthermia plus PBS injection). However, among those

experimental retinas, NMDA alone or preceded by hyperthermia did not significantly alter the number or optical density of HSP72-positive cells. Systemic administration

Table 1 Counts of TUNEL-positive nuclei and HSP72-positive cells in RGCL of hyperthermia pre-conditioned retinas 18 h after NMDA injection Type of pretreatment / time (h)

Intravitreal injection

Number of TUNEL-positive nuclei in RGCL

Number of HSP72-positive cells in RGCL

Number of retinas (n)

2/2 2/2 HT / 0 HT / 6 HT / 12 HT / 18 HT1Q / 12 HT / 0 HT / 6 HT / 12 HT / 18

– NMDA NMDA NMDA NMDA NMDA NMDA PBS PBS PBS PBS

0a 38.067.1 82.665.0 a 13.461.9 a 10.961.6 a 23.268.6 62.3620.0 a 0a 0.7560.5 a 0.2560.25 a 0.2560.25 a

33.063.4 47.468.4 b 48.064.8 b 47.263.7 b 52.464.6 b 42.364.8 b 34.366.2 50.667.7 b 39.565.6 b 52.463.2 b 48.066.2 b

5 7 5 5 5 6 6 5 4 4 4

HT represents hyperthermia; Q represents systemic administration of quercetin. The number of counted cells was averaged from two paraffin sections for one retina. Data are expressed as mean6S.E.M. Time ‘0’ represents that intravitreal injection was performed immediately after hyperthermia. a P,0.05, compared with non-pre-conditioned retinas after NMDA injection. b P,0.05, compared with normal control retinas.

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of quercetin reduced the number of HSP72-positive cells in the RGCL (Table 1). In contrast, there was no apparent difference in HSC70 immunoreactivity in retinas with NMDA injection and / or hyperthermic pre-conditioning compared with normal controls. No noticeable change of HSP72 or HSC70 was detected in other retinal layers (data not shown; at least five retinal sections per group were examined).

3.4. Neuronal survival in NMDA-excitotoxicity after hyperthermic pre-conditioning Flat preparations of retinas obtained 7 days after NMDA injection showed a loss of RGC-like neurons (Fig. 6D). Hyperthermic pre-conditioning given 6 or 12 h before NMDA injection appeared to ameliorate this loss showing more cells than the one without pre-condition (Fig. 6E). When these cells were counted in the central, midperipheral and peripheral retinas of animals, significantly more (P,0.05) RGC-like neurons were recorded in the 6 or 12 h pre-conditioned NMDA-injected retinas than in the corresponding areas of the NMDA-injected retinas that were not pre-conditioned (Fig. 7). However, when hyperthermic pre-conditioning was given 18 h before or simultaneously with NMDA injection, there was no ameliorative effect (P.0.05). The protection offered by hyperthermic pre-conditioning 12 h before NMDA injection was abolished by the simultaneous administration of quercetin.

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4. Discussion The present study demonstrates that intravitreal NMDA injection alone or hyperthermic pre-conditioning induces HSP72 but not HSC70 in the RGCs of rat retinas. Maximal levels of HSP72 in the RGCL were reached between 12 and 24 h after NMDA injection. A majority of surviving TUNEL-negative RGCs expressed HSP72, whereas only a few HSP72-positive RGCs were TUNEL-positive. Furthermore, hyperthermia induced 6 or 12 h before NMDA injection reduces the number of TUNEL-positive cells in the RGCL and ameliorates the neuronal loss caused by NMDA-induced excitotoxicity. Quercetin administration abolished the hyperthermic pre-conditioning-mediated protection of neurons, induction of HSP72 and inhibition of apoptosis-like cell death. These findings are consistent with a neuroprotective role of HSP72 in NMDA-induced RGC cell death. Consistent with the findings of Dean et al. [13], who showed an abundant amount of HSC70 specifically in the RGCL of normal retinas, we also noted moderate to strong immunoreactivity of HSC70 in normal rat retinas. Although the expression of HSP72 has been shown to protect cerebral neurons from glutamate-mediated cell death by various investigators [31,49,8], whether the induction of HSP72 is specific to excitotoxic insults remains controversial. Our observation that HSP72, but not HSC70, in RGCs is elevated after NMDA injection and hyperthermia suggests that the two isoforms of the HSP70 family, HSP72 and HSC70, are differentially regulated and ex-

Fig. 7. Hyperthermic pre-conditioning on the number of surviving RGC-like neurons 7 days after NMDA injection. NMDA-induced cell losses in central (1 mm), midperipheral (2 mm), and peripheral (3 mm) retinas were significantly ameliorated by hyperthermic pre-conditioning 6 or 12 h but not 18 h before NMDA injection (*, † and ‡P,0.05 when compared with central, midperipheral and peripheral retinas of NMDA-treated group, respectively). Quercetin administration abolished the ameliorative effect of pre-conditioning at 12 h. H, hours prior to NMDA injection; HT, hyperthermia; Q, Quercetin. Values represent mean6S.E.M.

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pressed in response to NMDA-induced excitotoxicity as well as heat shock. It has been reported that under stressful conditions, the amount of constitutively expressed HSC70 in mammalian retinal cells may change less rapidly than HSP72 [2,13,31,49,8,15,1]. HSC70 is believed to maintain regular cellular functions, whereas HSP72, because it is highly inducible from a very low level, may play a more important role in the prevention of misfolding and aggregation of intracellular proteins under abnormal conditions [64,37]. A recent study demonstrated decreasing levels of HSC70 and its mRNA in aging monkey and human retinas, suggesting that HSC70 may contribute to susceptibility to age-related retinal diseases [5]. Consistent with earlier studies on primary cortical and hippocampal neuronal cultures after heat shock [62], we also noted different colocalization patterns of HSP72 and TUNEL in the RGCL. It is possible that the expression of HSP72 in response to NMDA-mediated apoptosis may involve complex mechanisms. However, since the majority of surviving RGCs (TUNEL-negative) was HSP72-positive, it is indicative of an elicited cellular response or cellular defense to excitotoxicity. Similar to other studies on neurons after heat stress injury [62] or focal cerebral ischemia [56], apoptotic cells in the RGCL were either only mildly HSP72 positive or not labeled at all. This may be because insufficient HSPs were synthesized to clear the continued production of abnormal polypeptides leading to apoptosis. It is also possible that HSP expression is linked to the differential susceptibilities of various subtypes of RGCs. Apoptotic cells expressing none or small amount of HSP72 may also represent an unsuccessful self-defense mechanism. Therefore, cells that fail to express enough HSPs become apoptotic and early induction of HSP72 in sufficient quantity may rescue neurons from apoptosis. Whether there are subpopulations of cells that can respond faster and / or in larger quantity with HSP72 synthesis, and hence are more resistant to cell death remains unclear. The ameliorative effect on cell death and the window of effectiveness (12 to 6 h prior to NMDA injection) of hyperthermic preconditioning observed in this study are consistent with the reported neuroprotective effect of hyperthermia in other cerebral neurons [61,33] and the temporal changes of RGCL HSP72 as reported by Tytell et al. [61] who showed that mRNA and protein levels of HSP72 in the RGCL of rat retinas increased 4 h after hyperthermia and then started to decline 18 h after hyperthermia. Our observations, that pre-conditioning at 0 or 18 h prior to NMDA failed to have a significant anti-apoptotic (lack of reduction in the number of TUNELpositive cells compared to the nor-preconditioned retinas) and a neuroprotective effect (no preservation of RGC-like cell numbers) while at 12 and 6 h were effective, suggest that sufficient levels of HSP72 in the early hours after NMDA injection are crucial for an effective anti-apoptotic action and hence neuroprotective action of HSP72. This is consistent with earlier reports [2,31,49].

Although HSP72 has been shown to be neuroprotective against various types of damages in cerebral neurons [11,25,45,28,65], this report demonstrates that HSP72 may play a role in the stress response to protect retinal neurons and that this protection may be specific to neurons in the RGCL. However, since our cell counts may include displaced amacrine cells in the RGCL, further studies are needed to examine whether the neuroprotective effect of elevated HSP72 is specific towards RGCs. The present study also demonstrates that the neuroprotective effect of hyperthermic pre-conditioning may be through anti-apoptotic mechanism as suggested by the inverse relationship between TUNEL-positive cells and final RGC-like cell counts in flat preparations of retinas. Overall, these data suggest that hyperthermic pre-conditioning may be applied within a therapeutic window and that the peak of HSP72 production in RGCs (4–18 h after hyperthermia) [61] may need to overlap with the apoptotic cascades from 0 to 18 h after NMDA injection, or that sustained or pulsed elevations of HSP72 are needed to protect retinal neurons from damage. While the neuroprotective effect of HSP72 seems well established, its mechanisms of actions are less clear. ¨ ¨ ¨ et al. [23] showed that HSP72 inhibited the Jaattela activation of cytosolic phospholipase A 2 and changes in nuclear morphology downstream of caspase-3. In contrast, other researchers [38,29] demonstrated that HSP72 overexpression prevented both DNA fragmentation and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), implying inhibition of apoptosis upstream of caspase-3. Recent studies showed that one of the primary sites of HSP72 inhibitory activity could lie downstream of the mitochondrial events and the release of cytochrome c [29,46]. Beere and Green [3] reviewed the literature and pointed out that HSP72 is an anti-apoptotic chaperone protein that may interfere at multiple stages of the apoptotic pathway [38,4,7,40,39]. These stages may include suppression of c-Jun N-terminal kinase (JNK) activation [38,39], prevention of cytochrome-c release [23,29], disruption of apoptosome formation [3,29,50], inhibition of apoptotic protease activating factor 1 (Apaf-1) oligomerization [50], and suppression of procaspase recruitment [3,50]. It is not yet clear whether there are preferential sites of action by HSP72 with different stress stimuli. Glaucoma can be considered a group of related neurodegenerative diseases and is characterized by typical patterns of visual field loss caused by the loss of RGCs and their axons [47,55,20] in which apoptosis is believed to play a role [24,41,59]. Wax and colleagues first demonstrated increased immunoreactivity of HSP27, a small-sized HSP, in RGCs and the optic nerve head in human glaucomatous eyes [60]. Caprioli and co-workers showed that there is an early induction of HSP72 in RGCs of rats with experimental glaucoma and that the induced synthesis of HSP72 by systemic stressors such as hyperthermia or intraperitoneal injection of zinc increases RGC survival in these animals

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[43]. Hence, enhancement of endogenous self-defense mechanisms could be a possible approach to rescuing neurons in glaucoma. Our study confirms the neuroprotective effect of hyperthermia against NMDA-induced excitotoxicity and its anti-apoptotic effect in RGCs. How HSP72 regulates apoptosis and the roles of other members of HSPs in apoptosis are not yet known [34]. However, it is highly likely that HSP72 plays a very important role in promoting the survival of RGCs in neurodegenerative diseases.

[14]

[15]

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Acknowledgements [19]

Supported in part by the Glaucoma Research Foundation, San Francisco, California (Dr Caprioli); Research to Prevent Blindness, New York, NY (Dr Caprioli); and in part by CUHK Direct Grant P2040554, CUHK (Drs Kwong and Lam). Commercial Relationships Policy: N.

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