Caspase-2 Mediates Site-Specific Retinal Ganglion Cell Death After Blunt Ocular Injury.

Purpose: Ocular trauma is common in civilian and military populations. Among other injuries, closed globe blunt ocular trauma causes acute disruption of photoreceptor outer segments (commotio retinae) and retinal ganglion cell (RGC) death (traumatic optic neuropathy [TON]), both of which permanently impair vision. Caspase-2-dependent cell death is important and evidenced in models of RGC degeneration. We assessed the role of caspase-2 as a mediator of RGC and photoreceptor death in a rat blunt ocular trauma model. Methods: Bilateral ballistic closed globe blunt ocular trauma was induced in female Lister-hooded rats and caspase-2 cleavage and localization assessed by Western blotting and immunohistochemistry. Retinal caspase-2 was knocked down by intravitreal injection of caspase-2 small interfering RNA (siCASP2). In retinal sections, RGC survival was assessed by BRN3A-positive cell counts and photoreceptor survival by outer nuclear layer (ONL) thickness, respectively. Retinal function was assessed by electroretinography (ERG). Results: Raised levels of cleaved caspase-2 were detected in the retina at 5, 24, and 48 hours after injury and localized to RGC but not photoreceptors. Small interfering RNA-mediated caspase-2 knockdown neuroprotected RGC around but not in the center of the injury site. In addition, caspase-2 knockdown increased the amplitude of the ERG photopic negative response (PhNR) at 2 weeks after injury. However, siCASP2 was not protective for photoreceptors, suggesting that photoreceptor degeneration in this model is not mediated by caspase-2. Conclusions: Caspase-2 mediates death in a proportion of RGC but not photoreceptors at the site of blunt ocular trauma. Thus, intravitreally delivered siCASP2 is a possible therapeutic for the effective treatment of RGC death to prevent TON.

Regulation of Cdc42 expression and signaling is critical for promoting corneal epithelial wound healing.

PURPOSE: Cdc42, a member of Rho GTPases (guanosine triphosphatases), participates in cytokine- and growth factor-controlled biological functions in mammalian tissues. Here, we examined Cdc42 role in corneal epithelial wound healing and the influence of hepatocyte, keratinocyte, and epidermal growth factor (HGF, KGF, and EGF)-mediated signaling on Cdc42. METHODS: Epithelial wounds were created on the corneas of live rabbits by complete debridement and in rabbit corneal epithelial primary cultures through scratch injury. Cdc42 expression in cultures was suppressed using Cdc42 siRNA. Cdc42 activation was determined by pull-down assays with PAK-agarose beads. Cdc42 expression was analyzed by immunoblotting and immunofluorescence. Association of Cdc42 with cell-cycle proteins was identified by immunoprecipitation. RESULTS: In rabbit corneas, significant increase in Cdc42 expression that occurred 2 to 4 days after the injury coincided with wound closure, and by 8 days the expression reached near basal levels. Silencing of Cdc42 expression in cultures caused inhibition of wound closure as a result of 60% to 75% decrease in epithelial migration and growth. HGF, KGF, and EGF increased Cdc2 expression, activation, and its phosphorylation on ser71. Inhibition of growth factor-mediated PI-3K signaling resulted in the downregulation of Cdc42 expression and its phosphorylation. Increased association of cell-cycle proteins p27(kip) and cyclin-dependent kinase 4 (CDK4) with Cdc42; and phosphorylated Cdc42 with plasma membrane leading edges was also observed in the presence of growth factors. CONCLUSIONS: Cdc42 is an important regulator of corneal epithelial wound repair. To promote healing, Cdc42 may interact with receptor tyrosine kinase-activated signaling cascades that participate in cell migration and cell-cycle progression.

Ocular aldehyde dehydrogenases: protection against ultraviolet damage and maintenance of transparency for vision.

Aldehyde dehydrogenase (ALDH) enzymes catalyze the NAD(P)(+)-dependent oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding acids. Some members of the ALDH superfamily of enzymes are abundantly expressed in the mammalian cornea and lens in a taxon-specific manner. Considered to be corneal and lens crystallins, they confer protective and transparent properties upon these ocular tissues. ALDH3A1 is highly expressed in the cornea of most mammals, with the exception of rabbit that expresses exclusively ALDH1A1 in the cornea. ALDH1A1 is present in both the cornea and lens of several animal species. As a result of their catalytic and non-catalytic functions, ALDH3A1 and ALDH1A1 proteins protect inner ocular tissues from ultraviolet radiation and reactive oxygen-induced damage. In addition, these corneal crystallins contribute to cellular transparency in corneal stromal keratocytes, supporting a structural role of these ALDH proteins. A putative regulatory function of ALDH3A1 on corneal cell proliferation has also been proposed. Finally, the three retinaldehyde dehydrogenases cooperatively mediate retinoic acid signaling during the eye development.

Lysophospholipids and lysophospholipase D in rabbit aqueous humor following corneal injury.

We previously found that lysophosphatidic acid (LPA)-like activity eliciting Cl(-) currents in Xenopus oocytes is increased in rabbit aqueous humor (AH) following corneal freeze wounds. The purpose of this study was to examine whether actual levels of LPA in AH from wounded eyes are higher than those from control eyes, and to determine the sources and enzymatic pathways of AH LPA in control and wounded conditions. Lysophospholipase D (lysoPLD) activity was measured by the enzymatic determination of choline following incubation of AH samples with exogenous lysophosphatidylcholines (LPCs). The molecular species compositions of LPA and LPC in fresh and incubated AH were determined by liquid chromatography-tandem mass spectrometry. A high, but similar activity of lysoPLD in the samples from both control and freeze-wounded eyes was detected. Its enzymatic properties resemble those of plasma lysoPLD, identified as autotaxin. Levels of LPCs, predominant substrates of lysoPLD in AH, were several times higher in the AH samples from injured eyes than those from the control eyes. Our results suggest that lysoPLD is constitutively released from corneal tissues and/or ciliary body into the AH, with no injury-induced increase in release following freeze-wounding. They also suggest that wound-induced increases in LPA-like biological activity are due to linoleoyl species-rich molecular composition in AH from wounded eyes. A possible mechanism of the altered molecular composition is an increase in the AH concentrations of LPCs, linoleoyl species of which are preferentially converted to corresponding unsaturated LPA by the constitutively active lysoPLD.

Phospholipase C/Protein Kinase C pathway is essential for corneal re-epithelialization induced by Ap(4)A.

PURPOSE: We have previously demonstrated the importance of P2Y(2) receptors in the corneal re-epithelialization effect triggered by diadenosine tetraphosphate (Ap(4)A). In addition, we have also shown the ERK1/2 and ROCK-I activation in Ap(4)A-wound repair response. Phospholipase C/Protein Kinase C (PLC/PKC) pathway activation has been suggested as a molecular mechanism of growth factors-modulated corneal cell migration and P2Y(2) agonists. Hence, the aim of this study is to investigate the role of PLC/PKC cascade in the modification of re-epithelialization rate triggered by Ap(4)A in an established corneal epithelial cell line (Statens Seruminstitut rabbit cornea [SIRC] cells). METHODS: In wounded confluent SIRC cell monolayers and in the presence or absence of Ap(4)A 100 µM, a group of PLC/PKC inhibitors (U73122 3 µM, Staurosporine 1 nM and Bisindolylmaleimide-I 10 µM) and activator (PDBU 1 µM) were assayed and the migration rate was evaluated. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with or without Ap(4)A, U73122, Staurosporine, Bisindolylmaleimide-I and PDBU. RESULTS: Pre-treatment of wounded SIRC cells with PLC/PKC inhibitors significantly diminished the Ap(4)A-stimulated cell migration rate. Furthermore, PLC/PKC inhibitors also reduced ERK1/2 phosphorylation and ROCK-I activation triggered by Ap(4)A. CONCLUSIONS: The present study shows the involvement of PLC/PKC pathway in the activation of ERK1/2 and ROCK-I downstream signal transduction pathways stimulated by Ap(4)A/P2Y(2) receptor during corneal epithelial wound repair.

Corneal re-epithelialization stimulated by diadenosine polyphosphates recruits RhoA/ROCK and ERK1/2 pathways.

PURPOSE: To investigate the role of ERK1/2 and RhoA/ROCK intracellular pathways in the modification of corneal re-epithelialization when stimulated by the diadenosine polyphosphates Ap(4)A and Ap(3)A. METHODS: In wounded confluent SIRC (Statens Seruminstitut rabbit cornea) cell monolayers and in the presence or absence of Ap(4)A or Ap(3)A 100 microM, a battery of P2 receptor antagonists and inhibitors of tyrosin kinases, MAPK, and cytoskeleton pathways (AG1478 100 microM, U0126 100 microM, Y27632 100 nM, and (-)-blebbistatin 10 microM; n = 8 each) were assayed. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with Ap(4)A and Ap(3)A (100 microM), with or without suramin, RB-2, U0126, and Y27632. The intracellular distribution of pERK and ROCK-I was examined in the presence of Ap(4)A or Ap(3)A (100 microM) with U0126 and Y27632 (100 nM). RESULTS: In the presence of Ap(4)A, U0126, Y27632, AG1478, and (-)-blebbistatin, reduced the migration rate compared to the effect of Ap(4)A alone (P < 0.0001, P < 0.001, P < 0.01, and P < 0.1 versus Ap(4)A, respectively). In the presence of Ap(3)A 100 microM, U0126 and Y27632 accelerated the migration rate when compared with the effect of Ap(3)A alone, whereas AG1478 and (-)-blebbistatin (P < 0.0001 versus Ap(3)A) slowed the migration rate. Western blot assays demonstrated that both dinucleotides activated the ERK1/2 pathway but only Ap(4)A activated the ROCK-I pathway. The intracellular distribution of pERK1/2 and ROCK-I reflected cross-talk between these two pathways. CONCLUSIONS: The activation of the Ap(4)A/P2Y(2) receptor, accelerates corneal epithelial cell migration during wound healing with the activation of MAPK and cytoskeleton pathways, whereas activation of the Ap(3)A/P2Y(6) receptor signals only the MAPK pathway.

Unusually resistant post-traumatic uveitis with high serum ACE--an occult ocular sarcoidosis?

PURPOSE: The initiation of acute anterior uveitis following non-penetrating trauma comprises a defined clinical entity although its pathophysiology is not yet completely understood. DESIGN AND METHODS: Case presentation. RESULTS: We present two cases of young boys in whom a first episode of resistant anterior uveitis developed following relatively minor non penetrating ocular injury. Further investigation revealed high levels of serum ACE in both cases. CONCLUSION: To the best of our knowledge there are no previous published reports implicating minor ocular trauma in precipitating a first episode of uveitis associated with high serum ACE. A better understanding of the pathophysiology may provide insight into alternative treatments.

Expression of matrix metalloproteinase 2 and 9 in experimentally wounded canine corneas and spontaneous chronic corneal epithelial defects.

PURPOSE: To determine matrix metalloproteinase (MMP) 2 and MMP 9 expression in acute and chronic experimentally wounded canine corneas and keratectomy samples from canine patients with spontaneous chronic corneal epithelial defects (SCCEDs). METHODS: Mechanical debridement was performed unilaterally in 25 healthy dogs for the acute wound study. Twenty-four hours (n = 8), 48 hours (n = 5), 72 hours (n = 3), or 1 week (n = 9) after wounding, the dogs were euthanized. Debridement was performed once weekly for 8 weeks for the chronic study (n = 8). Therapeutic superficial keratectomies (n = 16) were performed on SCCED patients. Gelatin zymography and immunohistochemistry were performed. RESULTS: Acute wounds showed upregulation of MMP 9 at all time points. At 7 days after wounding, values diminished markedly but remained elevated above those of unwounded controls. SCCED and chronic wound samples showed a significant increase in MMP 9 compared with controls but were less than that of acute wounds. There was no significant difference between chronic wounds versus SCCED samples. Fellow control eyes showed significant upregulation of MMP 9 in tandem with wounded eyes. There was no significant difference in values for MMP 2 in wounded corneas or SCCED compared with those of controls. Immunhistochemistry localized MMP 9 to predominantly the epithelium with some staining of keratinocytes and stroma. CONCLUSIONS: The dog exhibits similar MMP expression during corneal wound healing to that of other species. The lack of significant difference in MMP expression between SCCED and chronic wounds suggest that MMP 2 and 9 are not involved in the pathophysiology of SCCED and are more likely altered secondary to a chronic epithelial defect.

Expression of matrix metalloproteinases 2 and 9 in experimental corneal injury and fungal keratitis.

PURPOSE: Levels of matrix metalloproteinases (MMPs) can be modulated during corneal infection, but little is known about MMP profiles during fungal keratitis. The purpose of this study was to determine the effect of corneal trauma and immunosuppressive treatment on the expression kinetics of MMP-2 and MMP-9 during experimental keratomycosis. METHODS: Corneas of immunocompetent and cyclophosphamide-treated adult BALB/c mice were topically inoculated with 1 x 10 culturable units of Fusarium solani or mock-inoculated with or without superficial corneal scarification. Eyes were scored daily for disease severity and processed for zymography after 1.5 hours, 6 hours, 1 day, 4 days, or 8 days. Gelatinase activity was densitometrically quantitated and normalized to MMP-2 and MMP-9 controls. RESULTS: MMP-9 levels in nontraumatized eyes transiently increased at 6 hours after fungal exposure, but this increase was inhibited by cyclophosphamide treatment. Corneal injury significantly induced early MMP-9 expression that returned to baseline levels within 4 days. Cyclophosphamide pretreatment reduced and delayed MMP-9 after scarification. Fusarium exposure dampened the MMP-9 response to corneal trauma in immunocompetent and cyclophosphamide-treated animals. Ocular levels of MMP-2 were not affected by scarification, fungal exposure, or immunosuppressive treatment. CONCLUSIONS: Ocular MMP-9 levels, but not MMP-2 levels, increased soon after corneal injury. A similar, although muted, MMP-9 response occurs during early filamentous fungal keratitis, with a kinetic profile similar to corneal disease progression. The early stage of ulcerative keratitis may involve selective regulation of corneal matrix metalloproteinases, suggesting an initial opportunity for therapeutic intervention.

[The effect of carnosine on inflammatory processes during contusion of the eyeball].

The effect of dipeptide carnosine on proteolytic processes accompanying inflammation was investigated in lacrimal fluid of patients with eyeball contusion. Eye drops containing 5% carnosine in combination with traditional treatment reduced elastase-like activity in lacrimal fluid and increased effectiveness of therapy.

Expression and distribution of tissue transglutaminase in normal and injured rat cornea.

PURPOSE: To determine the expression and distribution of tissue transglutaminase (TG(C)) and extracellular matrix (ECM) proteins in rat cornea during epithelial wound healing. METHODS: Corneal epithelial defects were created in rat corneas, and TG(C) expression was examined by Northern blot analysis, in situ hybridization, and immunohistochemical staining after the injury. The presence of fibrinogen, laminin-1, nidogen/entactin, and type collagen was also determined immunohistochemically. RESULTS: TG(C) was expressed in normal corneas. During the early wound healing process, TG(C) mRNA expression was up-regulated and TG(C) immunoreactivity was predominantly expressed in the migrating epithelial cells. ECM proteins were also expressed in a similar pattern as TG(C). CONCLUSIONS: The sites and time course of TG(C) expression indicate that TG(C) probably plays a role in maintaining the homeostasis of the cornea and in promoting epithelial wound healing. The simultaneous expression of TG(C) and ECM proteins suggests that the ECM proteins probably operate in concert with TG(C) in corneal wound healing.

The effect of sodium hyaluronate on the expression of gelatinases in rabbit corneal epithelial wound healing.

PURPOSE: We investigated the effect of sodium hyaluronate (Na-HA) on the expression of gelatinases in a rabbit model with corneal epithelial defects. METHODS: Topical administration of Na-HA or phosphate-buffered saline (PBS) was carried out in the experimental eyes after surgical removal of the corneal epithelium. At 0, 6, 24, 48, and 72 hours after wounding, mRNA expression of 72 kDa type gelatinase (MMP-2), 92 kDa type gelatinase (MMP-9), and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) were analyzed by reverse transcription polymerase chain reaction in those corneas. In addition, gelatinolytic activities were investigated using gelatin zymography. RESULTS: The levels of constitutive expression of MMP-2 and TIMP-1 mRNA persisted, whereas MMP-9 mRNA in the PBS-treated side was expressed temporarily after surgical removal. In the Na-HA-treated side, at 6 hours after wounding, a much higher expression of MMP-9 mRNA was reproducibly observed compared with that in the PBS-treated side. In zymography, the levels of gelatinolytic activity corresponding to proMMP-9 were significantly higher in the Na-HA-treated side than in the PBS-treated side at 6 hours after wounding. CONCLUSIONS: These results suggest a novel participation of Na-HA in the expression of MMP-9 in rabbit corneal epithelial wound healing.

Differential expression of MT1-MMP (MMP-14) and collagenase III (MMP-13) genes in normal and wounded rat corneas.

PURPOSE: Several members of the matrix metalloproteinase (MMP) group have been identified in the rat cornea during corneal wound healing. The aim of the present study was to identify additional members of the MMP gene family in the rat cornea and localize the expression of membrane type-1 matrix metalloproteinase (MT1-MMP; MMP-14) and collagenase III (MMP-13) in normal and wounded corneas. METHODS: Adult rats underwent laser keratectomy on the right eye. Unwounded left eyes were normal controls. Corneas were collected and processed at different times post-wounding. Reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing were used to discover the MMP genes expressed in the corneas. In situ hybridization was performed to localize the mRNA expression of MMP-14 and MMP-13. RESULTS: MMP-13 mRNA was detected in epithelial cells of wounded corneas, but not in normal controls; MMP-14 was found in both normal and wounded corneas. MMP-14 mRNA was expressed predominantly in the stromal keratocytes and rarely in the basal epithelial cells in normal and wounded corneas. MMP-13 mRNA was localized exclusively to basal cells of the epithelium at the wounded area from 6 hours to 3 days after wounding. CONCLUSIONS: MMP-14 and MMP-13 expression in rat corneas parallels that of gelatinases A and B, respectively. MMP-13 may play an important role in the gelatinase B-associated proteolytic cascade that allows rapid turnover of the extracellular matrix (ECM) components during corneal wound healing. MMP-14 may contribute to removing abnormal ECM components through activation of gelatinase A in rat corneas.

[Overall trypsin-like, elastase-like and antitryptic activity in patients with eye contusion]. / Obshchaia tripsinopodobnaia élastazopodobnaia i antitripticheskaia aktivnost' u patsientov s kontuziei glaza.

The investigations of tear fluid of eye after contusion injury revealed on increase of trypsin-like activity in 1-3 day and 10-19 day after trauma, the progressively elevation of elastase-like activity and lowering of inhibitory potential along 2-3 week. It was detected indirect correlation between the elastase-like activity and severity of the contusion injury of the eye, the grade of corneal edema, corneal erosion and conjunctival wound, hyphema. This results was shown the participation and the role of proteolytic and inflammatory processes in pathogenesis of eye blunt trauma. It was establish that the during of local inflammation is 2 week end more and is necessary antiinflammatory therapy with use the proteases inhibitors.

[The role of angiotensin-converting enzyme in pathogenesis of post-contusion disorders of ophthalmotonus and hemodynamics]. / Uchastie angiotenzinprevrashchaiushchego fermenta v patogeneze postkontuzionnykh narushenii oftal'motonusa i gemodinamiki.

Angiotensin converting enzyme (ACE) activity was studied in tear fluid of the contusion injured eye. We found substantial decrease of ACE activity during 1 month after trauma. Reduced ACE activity can potentiate kinin action and may contribute to the maintenance of reduced vascular tone, high capillary permeability inducing ciliochoroidal effusion which leads to ocular hypotony. We have found decrease of ACE activity in another healthy eye.

[The effect of Hirudo therapy on a change in the activity of retinal transport adenosinetriphosphatases in the rabbit with photic eye damage]. / Vliianie girudoterapii na izmenenie aktivnosti transportnykh adenozintrifosfataz setchatki krolika pri svetovom povrezhdenii glaz.

Effect of Hirudo therapy on the activities of transport adenosine triphosphatases (ATPases) of the retina and pigmented epithelium (PE) is studied in normal and albino rabbits whose eyes were exposed to potent illumination (100,000 Lux at the level of the cornea for 90 min). The exposure decreased the activities of ATPase, which did not recover in any of animal groups. Hirudo therapy immediately after illumination increased the enzymes activity in pigmented animals in comparison with intact control: by 20% in the retina and by 23% in PE; Mg-ATPase activity increased by 23% in the retina and decreased by 10% in PE. In subsequent 24 h, ATPase activities decreased, but in comparison with exposed retina the activity of Na,K-ATPase in the retina was 70% increased and in PE 78% increased; the activities of Mg-ATPase were increased by 33 and 8%, respectively. Complete recovery of ATPase activities was attained in 8 days. In albino animals, ATPase activities did not recover completely, but they were notably higher than in intact controls. Hirudo therapy before illumination had a marked protective effect.

Corneal epithelial wound healing increases the expression but not long lasting activation of the p85alpha subunit of phosphatidylinositol-3 kinase.

PURPOSE: Corneal epithelial wound healing is a complex process involving several growth factors whose interaction with tyrosine kinase receptors (RTK) leads to the recruitment of enzymes coupled to second messengers that propagate and amplify growth factor-induced signals inside the cells. Phosphatidylinositol-3 kinase (PI-3K) is one such enzyme. Here we have investigated changes in PI-3K activity and expression during re-epithelialization after in vivo and in vitro corneal injury. METHODS: For the in vivo model, epithelium was collected from rabbit corneas at different stages of wound healing after complete de-epithelialization. For in vitro studies, after 7 mm central scrape wounds were applied, rabbit corneas were maintained in organ culture. Immunoprecipitation and Western blot using anti-p85alpha antibodies were employed to determine PI-3K activity and expression of the p85alpha regulatory subunit of PI-3K. Two specific PI-3K inhibitors, Wortmannin and LY 294002 were used to study the effect of PI-3K activity on corneal epithelial wound healing. RESULTS: Two to four days after in vivo corneal epithelial wound healing, there was a 6-8 fold increase in the expression of the p85alpha subunit of PI-3K. By 8 days, the expression of p85alpha was similar to non-injured tissue. Increased expression of the 85kDa protein was observed mainly in the membrane fraction. Similarly, the expression of PI-3K was increased 24h after injured corneas were maintained in organ culture. Increase of p85alpha was confined to the wound region and surrounding area. No concomitant increase in PI-3K activity was observed in any of the wound models. Forty-eight hours after the central injury, Wortmannin and LY294002 inhibited wound healing by about 50%. CONCLUSIONS: Association of most of the increased p85alpha with the membrane fraction and no detectable increase in PI-3K activity during corneal re-epithelialization indicates that PI-3K activation is transitory. The results also suggest a mechanism of down regulation of the enzyme to avoid uncontrollable growth and cellular hypertrophy after growth factor stimulation during wound healing.

Corneal aldehyde dehydrogenase and glutathione S-transferase activity after excimer laser keratectomy in guinea pigs.

BACKGROUND: The free radical balance of the eye may be changed by excimer laser keratectomy. Previous studies have demonstrated that excimer laser keratectomy increases the corneal temperature, decreases the superoxide dismutase activity of the aqueous, and induces lipid peroxidation in the superficial corneal stroma. Aldehyde dehydrogenase (ALDH) and glutathione S-transferase (GST) are known to play an important role in corneal metabolism, particularly in detoxification of aldehydes, which are generated from free radical reactions. METHODS: In three groups of guinea pigs mechanical corneal de-epithelialisation was performed in group I, superficial corneal photoablation in group II, and deep corneal photoablation in group III, and the corneal ALDH and GST activities measured after 48 hours. RESULTS: The mean ALDH and GST activities of group I and II showed no differences compared with the controls (p > 0.05). The corneal ALDH activities were found to be significantly decreased (p < 0.05) and GST activities increased (p < 0.05) in group III. CONCLUSION: These results suggest that excimer laser treatment of high myopia may change the ALDH and GST activities, metabolism, and free radical balance of the cornea.

Ultrastructural findings and SOD levels in experimental optic nerve injury: effect of thyroid-releasing hormone on the degenerative and regenerative processes.

We performed pressure on the left optic nerves of 24 pigs and injected intravenous thyroid-releasing hormone (TRH) to 12 of these pigs in order to evaluate the degeneration and regeneration in the optic nerves. The histopathologic, ultrastructural and biochemical examinations of the optic nerves were made at the 24th hour, 7th, 15th and 30th days. Although the light-microscopic examinations were normal, ultrastructural changes of the uncompressed optic nerves were interesting. Histopathologic and ultrastructural investigation of the compressed optic nerves showed significant degenerative changes in the non-TRH-applied group. Ultrastructural comparison yielded lighter degenerative changes in the TRH-applied group but there was no clue showing the stimulation of regeneration. We observed increased superoxide dismutase (SOD) levels in the nontraumatized optic nerves due to the cellular stress. The SOD values were found to be low in highly damaged left compressed optic nerves indicating the prevention of SOD enzyme synthesis.

Increased gelatinolytic and caseinolytic activity in the thermally injured, nutritionally compromised rat cornea: detection of a 27-kDa lymphoreticular cell-associated caseinase.

This study assesses the impact of various forms of injury on matrix degrading enzymes in nutritionally compromised rat corneas. In vitamin A-deficient (nutritionally compromised) and normal control corneas, in vivo or ex vivo mild mechanical abrasion did not appreciably alter the activity of either the 65-kDa or the 92-kDa gelatinases. In contrast, after thermal injury, while no appreciable change was detected in activity associated with the 65-kDa gelatinase in either vitamin A-deficient or normal control corneas, 92-kDa gelatinolytic activity was consistently higher in corneas from both groups, although activity associated with nutritionally compromised corneas was much higher. In these corneas, thermal injury also induced the expression of two high molecular weight (approximately 130-kDa and 225-kDa) gelatinases and a 27-kDa caseinase. While gelatinases were totally inactivated by inhibitors of metalloproteinases such as 1,10-phenanthroline and Galardin MPI, the 27-kDa caseinase showed considerable susceptibility to a mixture of serine protease inhibitors (aprotinin, dichloro-isocoumarin and pA-PMSF [(4-amidino-phenyl)-methane-sulphonyl fluoride]. Furthermore, unactivated-lymphoreticular cells from either nutritionally compromised or normal control animals contained a 24- and 27-kDa caseinase, however most of the activity was due to the 24-kDa caseinase. In contrast, glycogen-activated lymphoreticular cells contained a preponderance of the 27-kDa caseinase. Activated-lymphoreticular cells also expressed 92-kDa, 130-kDa and 225-kDa gelatinases. The presence of low molecular weight caseinases in lymphoreticular cells implicates them as the source of these enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)