Tanya Myers

Injury to retinal ganglion cells induces expression of the small heat shock protein Hsp27 in the rat visual system.

Optic nerve transection results in apoptotic cell death of most adult rat retinal ganglion cells that begins at 4 days and leaves few surviving neurons at 14 days post-injury [Berkelaar et al. (1994) J. Neurosci. 14, 4368-4374]. The small heat shock protein Hsp27 has recently been shown to play a role in sensory neuron survival following peripheral nerve axotomy [Lewis et al. (1999) J. Neurosci. 19, 8945-8953]. To investigate the role of Hsp27 in injured CNS sensory neurons, we have studied the induction and cell-specific expression of Hsp27 in rat retinal ganglion cells 1-28 days after optic nerve transection. Immunohistochemical results indicate that Hsp27 is not present at detectable levels in the ganglion cell layer of control (uninjured) or sham-operated control rats. In contrast, Hsp27 is detected in retinal ganglion cells from 4 to 28 days following axotomy. Furthermore, the percentage of surviving retinal ganglion cells that are Hsp27-positive increased over the same time period. Hsp27 is also detected in glial fibrillary acidic protein-positive astrocytes in the optic layer of the superior colliculus from 4 to 28 days after optic nerve transection. These experiments demonstrate that transection of the optic nerve results in the expression of Hsp27 in three distinct regions of the rat visual system: sensory retinal ganglion cells in the eye, glial cells of the optic tract, and astrocytes in the optic layer of the superior colliculus. Hsp27 may be associated with enhanced survival of a subset of retinal ganglion cells, providing evidence of a protective role for Hsp27 in CNS neuronal injury.

Duration of lipid peroxidation after acute spinal cord injury in rats and the effect of methylprednisolone

OBJECT Oxidative stress leading to lipid peroxidation is a major cause of secondary injury following spinal cord injury (SCI). The objectives of this study were to determine the duration of lipid peroxidation following acute SCI and the efficacy of short-and long-term administration of methylprednisolone on decreasing lipid peroxidation. METHODS A total of 226 female Wistar rats underwent clip-compression induced SCI. In the first part of the study, spinal cords of untreated rats were assayed colorimetrically for malondialdehyde (MDA) to determine lipid peroxidation levels at various time points between 0 and 10 days. In the second part of the study, animals were treated with methylprednisolone for either 24 hours or 7 days. Control animals received equal volumes of normal saline. Treated and control rats were killed at various time points between 0 and 7 days. RESULTS The MDA levels initially peaked 4 hours postinjury. By 12 hours, the MDA levels returned to baseline. A second increase was observed from 24 hours to 5 days. Both peak values differed statistically from the trough values (p < 0.008). The methylprednisolone reduced MDA levels (p < 0.04) within 12 hours of injury. No effect was seen at 24 hours or later. CONCLUSIONS The results of this study indicate that oxidative stress persists for 5 days following SCI in rats, and although methylprednisolone reduces MDA levels within the first 12 hours, it has no effect on the second lipid peroxidation peak.

Administration of brain-derived neurotrophic factor suppresses the expression of heat shock protein 27 in rat retinal ganglion cells following axotomy.

Optic nerve transection results in the apoptotic cell death of the majority of retinal ganglion cells by 14 days. The neurotrophin brain-derived neurotrophic factor (BDNF) enhances survival of retinal ganglion cells. In addition, the small heat shock protein Hsp27, with its anti-apoptotic effects, may be important for neuron survival following axotomy or trophic factor withdrawal. We recently reported the induction and expression of Hsp27 in a subset of retinal ganglion cells following axotomy. Here we have examined the effect of BDNF administration on the expression of Hsp27 in axotomized adult rodent retinal ganglion cells. Retinal ganglion cells were pre-labeled with Fluorogold prior to optic nerve transection and concomitant intraocular injection of BDNF or vehicle. Hsp27 immunofluorescence was examined in retinal sections from 4 to 28 days following injury. Consistent with previous survival studies, the number of Fluorogold-labeled retinal ganglion cells declined from 100% at 4 days to approximately 15% by 14 days following axotomy and vehicle injection. In contrast, with BDNF administration, retinal ganglion cell survival was maintained at 100% to 7 days following axotomy. We report that the number of Hsp27-positive injured retinal ganglion cells, as detected by immunohistochemical staining, was decreased by 50% in BDNF-treated retinas, when compared with vehicle-treated controls. This decreased expression of Hsp27 in response to BDNF treatment was seen both at early (4 days) and delayed (14 days) times. BDNF following optic nerve transection significantly reduced the expression of Hsp27 in retinal ganglion cells. These results indicate that BDNF may down-regulate alternate cell survival pathways, including the stress-induced expression of Hsp27, and may help to explain the failure of chronic neurotrophin treatment to maintain long-term retinal ganglion cell survival.

Enriched environment during adolescence changes brain-derived neurotrophic factor and TrkB levels in the rat visual system but does not offer neuroprotection to retinal ganglion cells following axotomy

The purpose of the present experiment was to characterize changes in TrkB signaling in the rat visual system resulting from exposure to enriched environment. Female Sprague-Dawley rats were placed in enriched or impoverished conditions for 1, 7 or 28 days. Levels of BDNF protein and its predominant receptor TrkB were examined in the retina, superior colliculus and visual cortex. In the retina, 1 day of enrichment increased full-length TrkB and after 28 days increased BDNF. In the superior colliculus, enrichment for 7 days reduced full-length TrkB and after 28 days increased BDNF and full-length TrkB. One day of enrichment significantly increased BDNF, reduced full-length TrkB and increased truncated TrkB in the visual cortex. Consequently, we further investigated whether exposure to enriched environment and the subsequent changes in BDNF and TrkB translates into a neuroprotective effect on retinal ganglion cells (RGCs) following transection of the optic nerve. Although exogenous intraocular application of BDNF provides neuroprotection to RGCs after axotomy, the endogenous increase in BDNF in the retina after 28 days of enrichment had no effect on RGC survival. While enriched housing conditions offer a model of non-invasive rehabilitation treatment for injury and modulates changes in BDNF and TrkB levels, these molecular changes did not translate into a neuroprotective effect on RGCs following transection of the optic nerve.

Neural progenitor potential in cultured Müller glia: effects of passaging and exogenous growth factor exposure.

The Müller radial glial cell is the principal support cell of the adult mammalian retina. Recent reports suggest that these cells retain the capacity to proliferate, express phenotypes reminiscent of retinal progenitor cells (RPC) and generate neuron-like progeny. We isolated rodent Müller cells and generated cultures that could be passaged under conditions used in neural stem/progenitor cell colonies. We demonstrate that during the early period of primary culture, Müller glia proliferate into sphere colonies and express a select regimen of phenotypes normally seen in RPCs. This effect correlates temporally with the loss of retinal neurons post-dissection. When chronically maintained in vitro, Müller cells can be repeatedly passaged, and up-regulate early RPC phenotypes that are suggestive of cellular de-differentiation. Furthermore, exposure of Müller glial cultures to differentiating conditions containing growth factors stimulates Müller glia to up-regulate phenotypes associated with retinal neurons. These data provide further evidence that isolated, adult Müller glia retain functional and phenotypic features of RPCs.

Enhanced expression of heat shock protein 27 is correlated with axonal regeneration in mature retinal ganglion cells

The small heat shock protein, Hsp27, promotes axonal regeneration in peripheral neurons; however, an analogous role in the central nervous system has not been described. This study examined the relationship between Hsp27 expression and regeneration in mature retinal ganglion cells (RGCs). Adult rat optic nerves were transected and exposed to peripheral nerve autografts to stimulate regeneration of cut RGC axons. There was a five-fold increase in the Hsp27-positive fraction of RGCs that extended new axons into the graft when compared with those that survived injury but did not regenerate (30% versus 6% respectively, P = 0.001). Hsp27 protein was located throughout somata and neuritic processes, and there was a significant positive correlation between Hsp27 expression and axonal regeneration in injured neurons (R = 0.92, P < 0.0001). These findings are consistent with the growth-associated role of Hsp27 demonstrated in peripheral neurons and suggest that Hsp27 may mediate similar physiological functions in the central nervous system.

Expression of Hsp27 in retinal ganglion cells of the rat during postnatal development

The small heat shock protein Hsp27 has been shown to protect neurons from apoptosis. We have recently shown the expression of Hsp27 in a subset of injured adult retinal ganglion cells (RGCs), a response that is muted by the administration of brain‐derived neurotrophic factor. This work has suggested a role for Hsp27 in the long‐term survival of RGCs following injury. The purpose of this study was to investigate the expression of Hsp27 during postnatal retinal development, based on Hsp27s role as a neuronal survival factor and on its up‐regulation in the adult injured retina. Expression of Hsp27 in the developing retina was examined at various times postnatally (between P0 and P24) by using immunohistochemical techniques. We report that Hsp27 expression peaks in the ganglion cell layer between P6 and P12 and is not detected at earlier (P0–P3) or later (P15–P24) times. Double labeling of the Hsp27‐positive cells with Fluorogold applied to the superior colliculus confirmed that Hsp27‐positive cells in the ganglion cell layer are RGCs. We have shown developmentally regulated expression of Hsp27 in RGCs of the postnatal rat. The retinal expression of Hsp27 correlates temporally with innervation of the tectum by late‐born RGCs and with onset of spontaneous retinotectal activity. We propose that the expression of Hsp27 may play an important role in retinal development during a critical period of RGC functional connectivity with the superior colliculus. J. Comp. Neurol. 478:143–148, 2004.

Myocardial migration by fibroblast progenitor cells is blood pressure dependent in a model of angII myocardial fibrosis.

Activation of the renin–angiotensin system (RAS) is thought to promote myocardial fibrosis. However, it is unclear whether this physiological fibrotic response results from chronic hemodynamic stress or from direct cellular signaling. Male C57B/6 mice were randomly assigned to receive angiotensin II (AngII) (2.0 μg kg−1 min−1), AngII+hydralazine (6.9 μg kg−1 min−1) or saline (control) via osmotic pumps for 7 days. Blood pressure was measured via noninvasive plethysmography. Hearts were harvested and processed for analysis. Cellular infiltration and collagen deposition were analyzed using histological staining. Molecular mediators were assessed using quantitative RT-PCR. As previously described, animals that received AngII developed hypertension and multifocal cellular infiltration by SMA+/CD133+ fibroblast progenitors followed by collagen deposition. The coadministration of hydralazine with AngII completely inhibited the hypertensive effects of AngII (P⩽0.01) and resulted in minimal cellular infiltration and minimal collagen deposition. These findings were in the context of persistent RAS activation, which was evidenced by elevation in serum aldosterone levels in animals that received AngII or AngII+hydralazine compared with animals that received saline. At the molecular level, infusion of AngII resulted in the significant upregulation of profibrotic factors (connective tissue growth factor-7.8±0.7 fold), proinflammatory mediators (TNFα-4.6±0.8 fold; IL-1β-6.4±2.6 fold) and chemokines (CCL2-3.8±1.0 fold; CXCL12-3.2±0.4 fold), which were inhibited when hydralazine was also infused. We provide evidence that myocardial infiltration by fibroblast progenitor cells secondary to AngII and the resultant fibrosis can be prevented by the addition of hydralazine. Furthermore, the beneficial effects of hydralazine were observed while maintaining RAS activation, suggesting that the mechanism of fibrosis is blood pressure dependent.

Effects of epidermal growth factor and erythropoietin on Müller glial activation and phenotypic plasticity in the adult mammalian retina

Retinal Müller glia have received considerable attention with regard to their potential to function as quiescent retinal precursors. Various activation strategies induce characteristic features of retinal progenitor cells in Müller glia; however, these are often accompanied by hallmark features of reactive gliosis. We investigated the effects of an intravitreal injection of epidermal growth factor (EGF), a known mitogen, and erythropoietin (EPO) on activation and expression of developmental phenotypes within the adult retina. Using thymidine‐analogue labeling as well as immunocytochemical and confocal analyses, we assayed the responses of retinal cells exposed to intravitreal administration of either EGF or EPO. We report that adult Müller glia incorporate bromodeoxyuridine (BrdU) and undergo a process of nuclear translocation to ectopic retinal layers following exposure to EGF. These cells survive within the retina for at least 23 days and express the developmental markers Pax6 and Chx10 as well as nestin and glial fibrillary acidic protein. Furthermore, we demonstrate that cotreatment with EGF and EPO suppresses aspects of EGF‐induced glial reactivity, alters the retinal distribution of BrdU‐positive nuclei, and serves to regulate the expression of developmental phenotypes seen in these cells. These data further our understanding of Müller cell responsiveness to intravitral, combinatorial growth factor treatments.

Integrins and monocyte migration to the ischemic myocardium.

ABSTRACT Aims: Characterize mononuclear cell migration after acute-myocardial infarction (MI). Material and Methods: Male Lewis rats underwent a left thoracotomy and ligation of the left anterior descending coronary artery (MI group). Control animals underwent thoracotomy without ligation (Sham group). Animals were sacrificed at 0, 2, 4, or 24 hr after the onset of ischemia. Leukocyte migration was assessed using isolated and In111 labeled mononuclear cells (injected at the onset of ischemia) and γ-count determined at 24 hours. Inhibition of migration was evaluated with monoclonal anti α4 and/or β2 antibodies. Results: Serum troponin was significantly elevated in animals with MI as compared with Sham (p = .017). Labeled mononuclear cell migration was five-fold higher in MI-treated animals than in Sham (p = .006). ED-1 positive mononuclear cells were confirmed in the left myocardium after 24 hr of ischemia. MCP-1 mRNA was significantly elevated in the left myocardium at 2 hr and 4 hr and peaked at 24 hr (p <.05). In addition, α4 integrin blockade inhibited labeled mononuclear cell migration by 22%. Blockade of β2 integrin inhibited mononuclear cell migration by 48%, while the combined α4+β2 blockade resulted in 59% inhibition of labeled mononuclear cell migration compared with treatment with isotype control antibody (p = .001). Conclusions: Significant ED1+ mononuclear cell migration within 24 hr of MI correlated with peak MCP-1 mRNA. Monoclonal antibody blockade suggested that early mononuclear cell migration is dependent only in part on α4 and β2 integrins.