Nonlinear Inverted-U Shaped Relationship Between Aging and Epidermal Innervation in the Rat Plantar Hind Paw: A Laser Scanning Confocal Microscopy Study.

The under-reporting of pain and atypical manifestations of painful syndromes within the elderly population have been well documented, however, the specific relationship between pain and aging remains ambiguous. Previous studies have reported degenerative changes in primary afferents with aging. In this study, we questioned whether there is any change in the density of primary afferent endings within the epidermis of aged animals. Rats were categorically assessed in 4 age groups, each representing a key developmental stage across their life span: juvenile (2 months), adult (7 months); aged (18 months), and senescent (24-26 months). The plantar hind paw skin was removed, post-fixed, cut, and immunostained for protein gene product 9.5 and type IV collagen. Rats in the adult aged groups had significantly increased epidermal nerve densities and total lengths of immunoreactive nerve fibers, compared with juvenile as well as senescent rats. However, the paw withdrawal thresholds to punctate mechanical stimulation progressively increased with age, and did not exhibit a clear relationship with epidermal innervation. We conclude a nonlinear, inverted-U shaped relationship between rat plantar epidermal nerve density with aging, which does not correlate with mechanically-induced paw withdrawal behaviors. PERSPECTIVE: This article presents age-related decreased epidermal innervation in rat hind paw skin, which partly explains mechanisms underlying decreased pain sensitivity in aged subjects. The report may help clinicians to understand that any compromise of pain-sensing pathway can lead to under-reporting of pain, inadequate analgesia, and slower recovery from a painful condition.

Recombinant membrane-targeted form of CD59 inhibits the growth of choroidal neovascular complex in mice.

This study was designed to explore the effect of recombinant, membrane-targeted CD59 (rCD59-APT542) on the growth and size of fully developed neovascular complex using the murine model of laser-induced choroidal neovascularization (CNV). CNV was induced by laser photocoagulation in C57BL/6 mice using an argon laser, and the animals received rCD59-APT542 via intravitreal (ivt) route. Western blot analysis, immunohistochemistry, and total complement hemolytic assay demonstrated that exogenously administered rCD59-APT542 was incorporated as well as retained in RPE and choroid and was functionally active in vivo. Single ivt injection during the growth of the CNV (i.e. at day 3 post-laser) resulted in ∼79% inhibition of the further growth of neovascular complex. The size of the CNV complex was significantly (p < 0.05) reduced by the administration of rCD59-APT542 after the CNV complex has fully developed (i.e. at day 7 post-laser). Treatment with rCD59-APT542 blocked the formation of membrane attack complex (MAC), increased apoptosis and decreased cell proliferation in the neovascular complex. On the basis of results presented here we conclude that recombinant membrane targeted CD59 inhibited the growth of the CNV complex and reduced the size of fully developed CNV in the laser-induced mouse model. We propose that a combination of two mechanisms: increased apoptosis and decreased cell proliferation, both resulting from local inhibition of MAC, may be responsible for inhibition of CNV by rCD59-APT542.

Alcohol and nicotine consumption exacerbates choroidal neovascularization by modulating the regulation of complement system.

The objective of the present study was to investigate the effect of alcohol and nicotine consumption on the pathogenesis of choroidal neovascularization (CNV) in rats after laser-photocoagulation. Confocal microscopic analysis demonstrated an increase in CNV complex size in rats fed with alcohol (2.3-fold), nicotine (1.9-fold), and the combination of alcohol and nicotine (2.7-fold) compared with the control groups. Immunohistochemical analysis revealed that alcohol and nicotine consumption increased MAC deposition and VEGF expression in laser spots. Expression of CD59 by RT-PCR and Western blot was drastically reduced in the animals that were fed with alcohol, nicotine and alcohol and nicotine compared to those fed with water alone and this was associated with exacerbation of CNV.

Expression of adiponectin in choroidal tissue and inhibition of laser induced choroidal neovascularization by adiponectin.

The aim of this study was to investigate the role of adiponectin (APN) in a mouse model of laser induced choroidal neovascularization (CNV). We have shown by immunohistochemistry that the expression of APN, adiponectin receptor 1, adiponectin receptor 2 and T cadherin gradually increased from day 1 to day 7 post-laser in laser treated mice compared to controls. Recombinant APN (rAPN) was injected intraperitoneally (i.p., 25 microg/mouse) or intravitreally (2 microg/eye) in lasered mice. Another set of lasered mice received APN peptide via i.p. (75 microg/mouse) or intravitreal (30 microg/eye) route. Control mice received a similar treatment with PBS, control protein or control peptide after laser treatment. We found that in the i.p. and intravitreal injection of rAPN resulted in 78% and 68% inhibition respectively in the size of CNV complex compared to control mice. Similar results were observed when APN peptide was injected intravitreally or i.p. Treatment with rAPN or the peptide resulted in decreased levels of vascular endothelial growth factor. Thus, APN inhibited choroidal angiogenesis and may have therapeutic implications in the treatment of wet age related macular degeneration.

CD59, a complement regulatory protein, controls choroidal neovascularization in a mouse model of wet-type age-related macular degeneration.

We have shown that membrane attack complex (MAC) formation via the activation of the alternative pathway plays a central role in the laser-induced choroidal neovascularization (CNV). This study was undertaken to understand the role of a complement regulatory protein, CD59, which controls MAC assembly and function, in this model. CNV was induced by laser photocoagulation in C57BL/6 and Cd59a(-/-) mice using an argon laser. Animals from each group were sacrificed on day 1, 3, 5, and 7 postlaser. Retinal pigment epithelium-choroid-scleral tissue was examined to determine the incidence and size of CNV complex, and semiquantitative RT-PCR and Western blot analysis for CD59a was studied. Recombinant soluble mouse CD59a-IgG2a fusion (rsCD59a-Fc) protein was injected via i.p. or intravitreal routes 24 h before laser. Our results demonstrated that CD59a (both mRNA and protein) was down-regulated during laser-induced CNV. Cd59a(-/-) mice developed CNV complex early in the disease process. Increased MAC deposition was also observed in these Cd59a(-/-) mice. Administration of rsCD59a-Fc inhibited the development of CNV complex in the mouse model by blocking MAC formation and also inhibited expression of angiogenic growth factors. These data provide strong evidence that CD59a plays a crucial role in regulating complement activation and MAC formation essential for the release of growth factors that drive the development of laser-induced CNV in mice. Thus, our results suggest that the inhibition of complement by soluble CD59 may provide a novel therapeutic alternative to current treatment.

Complement activation via alternative pathway is critical in the development of laser-induced choroidal neovascularization: role of factor B and factor H.

The objective of this study was to explore the role of classical, lectin, and alternative pathways of complement activation in laser-induced choroidal neovascularization (CNV). The classical and alternative pathways were blocked in C57BL/6 mice by small interfering RNAs (siRNA) directed against C1q and factor B, respectively. C4(-/-) mice developed CNV similar to their wild-type controls and inhibition of C1q by siRNA had no effect on the development of CNV. In contrast, CNV was significantly inhibited (p < 0.001) in C5(-/-) mice and C57BL/6 mice treated with factor B siRNA. Inhibition of the alternative pathway by factor B siRNA resulted in decreased levels of membrane attack complex and angiogenic factors-vascular endothelial growth factor and TGF-beta2. Furthermore, factor B was up-regulated in complement sufficient C57BL/6 mice at day 1 postlaser and remained elevated at day 7. Significantly reduced levels of factor H were observed at day 3 in these animals. In conclusion, our results demonstrate that activation of the factor B-dependent alternative pathway, but not the classical or lectin pathways, was essential for the development of CNV in mouse model of laser-induced CNV. Thus, specific blockade of the alternative pathway may represent a therapeutically relevant strategy for the inhibition of CNV.

Alcohol linked to enhanced angiogenesis in rat model of choroidal neovascularization.

One of the pathologic complications of exudative (i.e. wet-type) age-related macular degeneration (AMD) is choroidal neovascularization (CNV). The aim of this study was to investigate whether chronic and heavy alcohol consumption influenced the development of CNV in a rat model. The oxidative metabolism of alcohol is minimal or absent in the eye, so that ethanol is metabolized via a nonoxidative pathway to form fatty acid ethyl esters (FAEE). Fatty acid ethyl ester synthase (FAEES) was purified from the choroid of Brown Norway (BN) rats. The purified protein was 60 kDa in size and the antibody raised against this protein showed a single band on western blot. BN rats on a regular diet were fed alcohol for 10 weeks. Control rats were fed water with a regular diet and pair-fed control rats were fed regular diet, water and glucose. We found that FAEES activity was increased 4.0-fold in the choroid of alcohol-treated rats compared with controls. The amount of ethyl esters produced in the choroid of 10 week alcohol-fed rats was 7.4-fold more than rats fed alcohol for 1 week. The increased accumulation of ethyl esters was associated with a 3.0-fold increased expression of cyclin E and cyclin E/CDK2; however, the level of the cyclin kinase inhibitor, p27Kip, did not change. The increased accumulation of ethyl esters was also associated with 3.0-fold decreased expression of APN in the choroid. We also found that the size of CNV increased by 28% in alcohol-fed rats. Thus, our study showed that chronic, heavy alcohol intake was associated with both an increased accumulation of ethyl esters in the choroid and an exacerbation of the CNV induced by laser treatment. These results may provide insight into the link between heavy alcohol consumption and exudative AMD.

Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization.

Choroidal neovascularization (CNV), or choroidal angiogenesis, is the hallmark of age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been explored experimentally. A reliable way to produce CNV in animals is to rupture Bruch's membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and membrane attack complex (MAC) in the neovascular complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3(-/-) mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-beta2, and beta-fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser- induced choroidal angiogenesis in mice. It is possible that a similar mechanism may be involved in the pathophysiology of other angiogenesis essential diseases.