Role Of Calpains In The Mechanism Of Ischemia/Reperfusion Injury In Rat Retina

Retinal ischemia is a common clinical entity which remains a common cause of visual impairment and blindness in the world because of the lack of effective treatment. Ischemia/reperfusion injury (IRI) of the retina is one of the deleterious pathophysiological processes leading to visual impairment. It is well studied that apoptosis and calcium concentration elevates in IRI. The family of calpains is one kind of neutral restrictive proteolytic enzyme which distributes diffusely. Under pathological condition, abnormal elevated calcium can activate calpains and induce diseases. E-64d is a specific inhibitor of calpains with high permeability and high combinative ability. It is known that E-64d can protect neural system from proteolytic enzyme.The retinal thickness and morphologic changes was detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was accessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain as well as calpastatin (an endogenous protein inhibitor of calpain) in the retina was accessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression calpain, the drug (5ul of 100uM) was injected intravitreously immediately after IRI. Our study showed that there were retinal edematous changes, particularly in the inner plexiform layer after IRI. Calpain is involved in the retina injury of rats induced by ischemia/reperfusion. Calpain inhibitor E-64d decreased the protein expression of calpain, as well as the ratio increase of calpain/calpastatin mRNA. E-64d also inhibited retinal damage induced by IRI, suggesting the possible role of E-64d in the protection of retina apoptosis induced by IRI..Role of Calpains in the Mechanism of High intraocular pressure (IOP)-induced Ischemia/Reperfusion Injury in Rat RetinaIntroductionRetinal ischemia is a common clinical entity which remains a common cause of visual impairment and blindness in the world because of the lack of effective treatment. Ischemia/reperfusion injury (IRI) of the retina is one of the deleterious pathophysiological processes leading to visual impairment. Understanding the pathophysiological effect of IRI on the retina is important for the development of therapeutic strategies for protecting the retinal neurons. Studies have suggested that IRI initiates apoptosis in the retinal neuro-epithelium.The cascade of neuronal apoptosis, as reported in other tissues, is evidenced by the increased expression of bcl-2 family genes such as bax (pro-apoptosis) and bcl-2 (anti-apoptosis), with an increased bax/bcl-2 ratio favoring apoptosis. The final 'execution' of apoptosis is mediated by the activation of cysteine proteases, including caspases and calpains. Calpains, which mainly consist of two isoforms named m-calpain(activated in mili-molar Ca²⁺) and u-calpain (activated in micro-molar Ca²⁺), which are regulated by the endogenous inhibitor, calpastatin. The activity of calpastatin is considered to play a critical role in preventing calpain-mediated catabolism in tissues. With an increased calpain to calpastatin ratio, calpastatin is digested by calpain and loses its inhibitory activity. Since calpain activity may mediate cell death and damage in the IRI, it is of considerable interest to explore the benefit of a therapeutic approach that involves calpain inhibitors in neurodegenerative diseases. It has already been shown that E-64d, the first reported cell permeable calpain inhibitor, is capable of providing potent neuro-protective effect by inhibiting calpain-related neuron apoptosis in the injury of spinal cord. However, the involvement of calpains, and further, the effect of E-64d, is still unclear in the retina neuronal apoptosis induced by IRI.High intraocular pressure (IOP)-induced ocular ischemia is a commonly used model for retinal ischemia research and has been described in a number of animal species, such as the rat and rabbit. High IOP induces global ischemia with obstruction of both the retinal and uveal circulation. This particular model presents pathological features which are almost identical to that seen after central retinal artery obstruction (CRAO). In such a model, we investigated the effect of E-64d on the expression of m-calpain and calpastatin in rat retina subjected to IRI. 1. Establishment of the Animal model of Ischemia/Reperfusion Injury in the Rat RetinaPurpose:To study the possibility of using high intraocular pressure (IOP)-induced Ischemia/Reperfusion Injury as the animal model.Methods:Sprague-Daley rats weighing 200-300g were purchased from the Shanghai laboratory animal center, the Chinese Academy of Science. Rats were maintained under standard lighting conditions with a 12:12h light/dark cycle at a room temperature between 21～24°C. A slit-lamp microscopic examination was performed on each rat to exclude any ocular defect. The rats were then anesthetized with 10% chloral hydrate (400 mg/kg) intraperitoneally. A 20-gauge needle connected with normal saline tubing was introduced into the anterior chamber of the right eye. A high IOP (109.7 mmHg) was achieved and maintained by lifting the infusion bottle to a height of 150 cm for 1 hour. The high IOP was attenuated by taking out the needle from the anterior chamber. The rat was then sacrificed the eyeball enucleated at 1,3,6,24 or 72 hours after the procedure. The eyeballs were fixed with paraformaldehyde and embedded in paraffin. The retinas were cut into 5μm thick sections and were processed for hematoxylin and eosin staining as well as immunohistochemistry.Results:There was an obvious increase in retinal thickness after IRI, particularly in the inner plexiform layer. Edematous changes were also seen in cells in the ganglion cell layer (GCL). Many pyknotic cells were found in the inner nuclear layer (INL) and outer nuclear layer (ONL) at 6h and 24h. By the end of the experiment, 72h after the IRI, cells in the retinal layers did exhibit obvious degenerative changes. Immunostaining of m-calpain as well as bax were detected slightly or not at all in GCL and INL. The staining became more intense from 1 to 24h, and was largely confined to the cells of the GCL and INL. There was most intense staining in cells located in the outer regions of the INL. The staining in the INL appeared to mark a structure lining the outer boundary of this cellular layer and the outer plexiform layer.Conclusions:High intraocular pressure (IOP) can induce Ischemia/Reperfusion Injury in the rat retina. The changes of retinal morphology were similar with retinal ischemia. It is a recommendable way to use high intraocular pressure (IOP)-induced animal as the model of Ischemia/Reperfusion Injury. 2.Role of Calpains in the Mechanism of High intraocular pressure (IOP)-induced Ischemia/Reperfusion Injury in Rat RetinaPurpose:To investigate the role of apoptosis and Calpains expression in the high intraocular pressure (IOP)-induced Ischemia/Reperfusion Injury in Rat Retina. Methods: Animal model retina IRI was set up by increasing the intraocular pressure (110 mmHg) of a rat eye for 1 h. The retinal thickness and morphologic changes was detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was accessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain as well as calpastatin (an endogenous protein inhibitor of calpain) in the retina was accessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated.Results:There were retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24h after IRI. The mRNA expression of m-calpain and calpastatin was also increased 24h and 3h after IRI, respectively. The mRNA ratio of mcalpain/calpastatin was increased at the 6h, 24h and 72h after IRI (P < 0.05).Conclusions:There were apoptosis and increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain/calpastatin in the rat retina of IRI. 3. Inhibition of Calpain Expression by E-64d in the Rat Retina Subjected to Ischemia/Reperfusion InjuryPurpose:To investigate the effect of E-64d, a selective inhibitor of calpain, on the expression of calpain and calpastatin in rat retina of ischemia/reperfusion injury (IRI).Methods:Animal model of retina IRI was set up by increase the intraocular pressure (110 mmHg) of a rat eye for 1 h. The retinal thickness and morphologic changes was detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was accessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain as well as calpastatin (an endogenous protein inhibitor of calpain) in the retina was accessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression calpain, the drug (5 ul of 100 uM) was injected intravitreously immediately after IRI.Results:There was retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24h after IRI, an effect that was inhibited by E-64d (P < 0.05). The mRNA expression of m-calpain and calpastatin was also increased 24h and 3h after IRI, respectively. Neither m-calpain nor calpastatin mRNA expression was influenced by E-64d (P > 0.05). The mRNA ratio of mcalpain/calpastatin was increased at the 6h, 24h and 72h after IRI and only at 24h the increase of the ratio was inhibited by E-64d (P < 0.05). Conclusions:In the rat retina of IRI, E-64d inhibits the increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain/calpastatin. E-64d also inhibited retinal damage induced by IRI, suggesting the possible role of E-64d in the protection of retina apoptosis induced by IRI...