Stefan Isenmann

Proliferative response of microglia and macrophages in the adult mouse eye after optic nerve lesion.

PURPOSE The purpose of this in vivo study was to evaluate the proliferative response of immunologic cells during the acute phase after optic nerve (ON) lesion in the neural retina and the ciliary body (CB) in the adult mouse. METHODS The number of cells obtained 5 to 10 days after ON crush was compared with that counted after intraorbital ON transection. In addition, after ON crush, the time course of in situ proliferating Ki67(+) microglia and macrophages was analyzed from 6 hours up to 10 days. RESULTS The number of BrdU(+)F4/80(+) retinal microglia and ciliary macrophages increased over time, reaching the peak number 10 days after ON lesion. In the retina, both ON lesion types resulted in a similar number of BrdU(+)F4/80(+) microglia. Approximately 85% of all BrdU(+) cells were identified as F4/80(+) microglia. However, this cell population represented only 30% of all F4/80(+) microglia. The peak of microglial in situ proliferation was found 2 days after ON crush. In the CB, both ON lesion types induced a significant increase in the number of BrdU(+)F4/80(+) macrophages. Of interest, the number of cells after ON transection further increased over time, whereas those after ON crush did not. CONCLUSIONS ON lesion significantly increased proliferation of F4/80(+) immunologic cells in both the retina and CB. Although no significant differences in cellular response were observed in the retina between both lesion types, ON transection had a more pronounced effect on ciliary macrophages than did ON crush. Therefore, both regions seem not to act in concert during the acute phase after ON lesion.

Excess Bcl-XL increases the intrinsic growth potential of adult CNS neurons in vitro

The regenerative potential of adult mammalian CNS neurons is limited. Recent data suggest that inactivation of major growth inhibitors may not suffice to induce robust regeneration from mature neurons unless the intrinsic growth state is modulated. To investigate a possible role of Bcl-XL for axon regeneration in the adult mammalian CNS, Bcl-XL was adenovirally overexpressed in severed rat RGCs. Bcl-XL overexpression in mature axotomized RGCs in vivo increased both numbers [3.10-fold (+/-0.20)] and cumulative length [6.72-fold (+/-0.47)] of neurites regenerated from retinal explants, and this effect was further pronounced in the central retina where specific and dense axoplasmatic transduction occurs. Similarly, delayed Bcl-XL gene transfer to explanted retinae 12-13 days after lesion increased the numbers and length of emanating neurites by a factor of 5.22 (+/-0.41) and 8.29 (+/-0.69), respectively. In vivo, intraretinal sprouting of unmyelinated RGC axons into the nerve fiber layer was increased. However, fiber ingrowth into the optic nerve remained sparse, likely due to myelin inhibitors and scar components. Therefore, Bcl-XL overexpression may enhance, but may not be sufficient to, restitute functional regeneration in the adult CNS. As assessed by cell quantification analysis, Bcl-XL overexpression rescued a higher proportion of RGCs in vivo than in vitro. Therefore, Bcl-XL is capable to induce both neuronal survival and axon regeneration, but these two processes appear to be differentially modified by distinct pathways in vivo.

Optic nerve lesion increases cell proliferation and nestin expression in the adult mouse eye in vivo.

In the naïve adult rodent eye cell proliferation does not occur. The aim of this in vivo study was to evaluate if quiescent putative progenitor-like cells within the adult mouse eye can be activated by optic nerve (ON) injury. For a comprehensive analysis, three areas were assessed: the ON, the neural retina, and the ciliary body (CB). Two lesion types were performed, i.e. intraorbital ON transection, or ON crush lesion, in order to analyse possible differences in cellular response after injury. This mouse study shows, for the first time that ON lesion up-regulates cell proliferation and nestin expression in the mouse eye as compared to naïve controls. Numbers and distribution patterns of BrdU+ cells obtained were similar after both lesion types, suggesting analogous mechanisms of activation. Interestingly, a differential cell proliferative response was observed in the CB. After ON lesion, the absence of BrdU/TUNEL co-labelled cells confirmed that BrdU+ cells were indeed proliferating. Following ON lesion, in the retina approximately 18% of all BrdU+ cells were positive for the neural stem cell/progenitor cell (NSC/PC) marker nestin. The fraction of BrdU+/nestin+ cells in the CB was approximately 26%. Most of the BrdU+/nestin+ cells found in the neural retina were identified as reactive astrocytes and Müller cells. Since reactive glia cells can participate in adult neuro- and gliogenesis this may indicate a potential for regeneration after ON lesion in vivo.

Statins Modulate Heat Shock Protein Expression and Enhance Retinal Ganglion Cell Survival after Transient Retinal Ischemia/Reperfusion In Vivo

PURPOSE To evaluate putative mechanisms for the pleiotropic effects of statins, the expression of members of the heat shock family of proteins (HSPs) was compared between normal and ischemic rat retinas after transient retinal ischemia/reperfusion and statin treatment in vivo. METHODS Retinal ischemia/reperfusion was induced by transient elevation of intraocular pressure (IOP). Retinal expression of HSPs was evaluated at different time points after drug and solvent injection and retinal ischemia/reperfusion by means of PCR and Western blot analysis. Immunofluorescent staining and confocal laser scanning microscopy were used to localize the expression of HSPs in normal and ischemic retinas. RESULTS During the acute phase after retinal ischemia, alphaB-crystallin protein and mRNA expression were reduced after statin treatment. After 72 hours of reperfusion, statins increased the expression of alphaB-crystallin and reduced the expression of HSP27 in the retina. Increased expression of alphaB-crystallin early after lesion and statin delivery correlated with increased expression of the heat shock factors 1 and 2. Statins significantly enhanced retinal ganglion cell (RGC) survival 10 days after transient retinal ischemia in vivo. CONCLUSIONS Systemic delivery of statins after a transient period of retinal ischemia significantly modulated HSP expression in the retina and enhanced RGC survival. Together, these results support the notion that statins constitute a feasible therapeutic approach to prevent some of the neuronal damage in the acute and possibly also the delayed phase and have beneficial effects in central nervous system (CNS) disorders directly affecting the visual system.

Regulation of GDNF and its receptor components GFR-α1, -α2 and Ret during development and in the mature retino-collicular pathway

Abstract The development of the retino-tectal projection as part of the central visual pathway is accomplished around postnatal day (P) 10–14 in rodents, and trophic factors are important for topographic refinement of this projection. Emerging data indicate that GDNF may influence synaptic plasticity of this projection. To date, maturation-dependent kinetics of GDNF release and expression and biological function of single GDNF receptors along the retino-collicular pathway are ill-defined. Here, we examined mRNA and protein expression of GDNF and its multicomponent receptor complex in the retina and superior colliculus (SC) during postnatal development of the rat visual system, and after optic nerve (ON) injury by RT-PCR, immunoblotting and immunofluorescence. Stable mRNA transcription of GDNF and its receptors GFR-α1, -α2 and Ret was found in retina and SC throughout development into adulthood and after ON transection. Expression of GDNF protein increased during retinal development, declined in adulthood and was further reduced in injured retina. In the SC, GDNF peaked at P0, continuously declined with maturation, and was undetectable in the deafferentiated SC. GFR-α1 was abundant in retina and SC throughout, while GFR-α2 was not expressed. Since Ret was localized primarily to the vascular compartment, the receptor tyrosine kinase may play a minor role in neuronal GDNF signaling. In summary, we provide evidence for GDNF as survival and guidance factor during development of the retino-tectal projection with differential regulation in early and premature retina and SC. Postlesionally, midbrain targets do not induce GDNF, suggesting that retrograde GDNF is not essential for rescue of adult injured retinal ganglion cells (RGCs).

Impaired development of the Harderian gland in mutant protein phosphatase 2A transgenic mice

Although Harderian glands are especially large in rodents, many features of this retroocular gland, including its development and function, are not well established. Protein phosphatase 2A (PP2A) is a family of heterotrimeric enzymes expressed in this gland. PP2A substrate specificity is determined by regulatory subunits with leucine 309 of the catalytic subunit playing a crucial role in the recruitment of regulatory subunits into the complex in vitro. Here we expressed an L309A mutant catalytic subunit in Harderian gland of transgenic mice. We found a delayed postnatal development and hypoplasia of the gland, causing enophthalmos. To determine why expression of the L309A mutant caused this phenotype, we determined the PP2A subunit composition. We found an altered subunit composition in the transgenic gland that was accompanied by pronounced changes of proteins regulating cell adhesion. Specifically, cadherin and beta-catenin were dramatically reduced and shifted to the cytosol. Furthermore, we found an inactivating phosphorylation of the cadherin-directed glycogen synthase kinase-3beta. In conclusion, the carboxy-terminal leucine L309 of the PP2A catalytic subunit determines PP2A heterotrimer composition in vivo. Moreover, our data demonstrate that PP2A subunit composition plays a crucial role in regulating cell adhesion and as a consequence in the development of the Harderian gland.

A novel primary culture technique for adult retina allows for evaluation of CNS axon regeneration in rodents.

Unraveling the causes of regeneration failure in the adult injured CNS has remained a challenge in neurobiology. The notion that CNS neurons lose their regenerative potential during development has been challenged by the identification of several promoters of axon growth. Novel methods are required that allow to study and quantify interactions of molecular determinants, and to envisage future treatment applications. Here we report a novel, highly reproducible method for monitoring axonal regeneration of mature retinal ganglion cells (RGCs) in vitro. In contrast to earlier explantation methods, primary cultures derived from adult rodent retina are kept viable without growth factor supplements. Further, since intraretinal RGC axons remain unmyelinated, regeneration can be followed independently of non-permissive white matter compounds. Applying tracing techniques prior to retinal explantation, cell survival can be correlated to outgrowth activity on the single cell level. Following intervention with pharmacological, growth factor, or gene transfer treatments, retinal explants, and partially RGC neurites, can be processed for protein and gene expression analysis. This novel procedure will prove useful to get insight into complex cell survival and regeneration promoting cascades, and will complement in vivo strategies such as transgenic and knock out mouse models.

A primary culture technique of adult retina for regeneration studies on adult CNS neurons.

This protocol details a tissue culture technique that allows for quantified regeneration studies on adult retinal ganglion cells (RGCs), that is, CNS neurons. The method may also allow for elucidation of molecular cues, for example of signals relevant in neuronal survival and axon regeneration. The procedure relies on fractioned stripe culture of previously injured retina in defined culture media. Naive dendritic cell contacts of RGCs are preserved, and the system is independent of growth factors. In contrast to other techniques, the protocol is based on tissue grown from adult animals; it dispenses immature co-cultures and evaluates the outgrowth of unmyelinated neurites in a milieu lacking CNS myelin. The technique is suitable for rodent retina from mouse or rat. A growth-conditioning injury of the optic nerve is set 10 days before retinal explantation. Explants are cultured for 5–7 days. Mere preparation of a single retina should be completed within 20 min.

Dissections of Cervical Arteries – Clinical Presentation, Course, and Therapy in 71 Consecutive Patients of a Single University Centre

Dissections of the cervical arteries are among the most frequent causes of juvenile strokes. The etiology and pathogenesis of spontaneous dissections remain elusive. Best treatment remains to be defined. Here, we analyzed 71 consecutive patients from the Department of Neurology, University Hospital of Jena. We asked if immediate anticoagulation or alternative treatment with ASA would affect outcome. Patients treated initially with i.v. ASA tended to have a better outcome than patients who were anticoagulated (r=0.3; p<0.05). In heparin treated patients, an initial i.v. bolus shortened the interval before the target PTT was reached by 1.3 days (p<0.05), yet did not affect neurological outcome. Low NIHSS (National Institute of Health Stroke Scale) (r=-0.71; p<0.01) and high Barthel scores (r=0,77; p<0.01) at presentation predicted a good outcome. In 14 of 52 patients, low TSH (thyroid- stimulating hormone) indicated hyperthyreosis, while no patient was hypothyreotic. In 33 of 64 patients CRP (C-reactive protein) was elevated. These findings merit validation in larger trials.

A Swelling of the Right Neck and Sudden Death

A 56-year-old, previously healthy man presented with a painless pulsatile tumor of the right neck (Figure 1A). The remaining clinical and neurological examination was unremarkable. Duplex sonography, CT angiography, and digital substraction angiography revealed large aneurysms of both common and internal carotid arteries (Figure 1B and 1C). Aneurysms of the brachiocephalic and both subclavian arteries were also seen. An abdominal CT showed aneurysms of the infrarenal aorta (O 4.8 cm), the superior mesenteric, and both common iliac arteries (Figure 2A). A chest CT was not performed. However, on reevaluation of the abdominal CT three months later, the uppermost section showed the right coronary artery with a diameter of 33 mm (Figure 2B and 2C) …