Harald D. Müller

Neuroprotection by Hyperbaric Oxygenation After Experimental Focal Cerebral Ischemia Monitored by MRI

Background— Hyperbaric oxygenation (HBO) after focal cerebral ischemia reduces infarct size and improves outcome when applied early after stroke. Here, we evaluated effects of HBO on permanent focal cerebral ischemia and applied magnetic resonance imaging (MRI) monitoring to study lesion evolution. Methods— Rats underwent permanent middle cerebral artery occlusion (MCAO). Two hours later, animals were treated with HBO (100% O2/2 atm; n=17) for 1 hour or treated with room air (n=17). Animals underwent serial MRI studies (DWI, PI, T2) beginning 90 minutes after MCAO. Neuroscore was assessed (5-point rating scale). Animals were euthanized and brains were 2,3,5-triphenyltetrazolium chloride (TTC)-stained for infarct volume calculation 120 hours after MCAO. Immunohistochemistry was performed with antibodies against c-FOS and 4-hydroxy-2-nonenal-modified proteins (HNE) to check for effects of oxidative stress caused by HBO treatment. Results— HBO reduced infarct volume by 38% (P <0.001). As shown by MRI, neuroprotection began 5 hours after ischemia and remained effective for 5 days. The relative regional cerebral blood flow was not different between groups at 3.5 and 5 hours after occlusion. There was less neurological deficit in HBO-treated animals compared with controls (P <0.05). Lipid peroxidation of cerebral vessels after HBO treatment as measured by HNE staining and pattern of c-FOS induction were not significantly different between groups at 3.5 and 8 hours after ischemia. Conclusion– As monitored by MRI HBO treatment reversed ischemic lesion size between 3 and 5 hours after ischemia and achieved a long-lasting neuroprotective effect without significant oxidative damage.

Brain-Derived Neurotrophic Factor But Not Forced Arm Use Improves Long-Term Outcome After Photothrombotic Stroke and Transiently Upregulates Binding Densities of Excitatory Glutamate Receptors in the Rat Brain

Background and Purpose— Both application of neurotrophic factors like brain-derived neurotrophic factor (BDNF) and constraint-induced movement therapy like forced arm use have been shown to potentially improve outcome after stroke. The aim of the present study was to check whether postischemic long-term outcome correlates to specific modifications in the abundance of various neurotransmitter receptors. Methods— Adult male Wistar rats were subjected to photothrombotic ischemia and assigned to various treatment groups (n=5 each) with end points at 3 and 6 weeks: (1) ischemic control (saline); (2) BDNF (ischemia, 20 &mgr;g BDNF); (3) forced arm use (ischemia, saline, and ipsilateral plaster cast for 5 or 14 days for the 3- and 6-week groups, respectively); and (4) combined treatment (combi; ischemia, 20 &mgr;g BDNF, forced arm use). Animals received intravenous bolus infusions of saline or BDNF 1 hour 3 and 5 days after ischemia, respectively. A group of sham rats (n=2) served as a control. A battery of behavioral tests was performed before and up to 6 weeks after ischemia. Quantitative in vitro receptor autoradiography was performed on 12-&mgr;m-thick cryostat sections using [3H]MK-801, [3H]AMPA, and [3H]muscimol for labeling of NMDA, AMPA, and GABAA receptors, respectively. Results— Best functional outcome was seen after BDNF treatment, whereas vice versa rats with forced arm use did worse in behavioral performance. Improved behavioral outcome was associated with increased perilesional binding densities of NMDA and AMPA receptors 3 weeks after stroke. Conclusions— Our findings suggest that transient enhanced neurotransmission as reflected by increased ligand binding of NMDA and AMPA receptors may participate in successful postlesional reorganization processes.

Endocannabinoids mediate neuroprotection after transient focal cerebral ischemia

The endocannabinoids anandamide (AEA) and palmitoylethanolamide (PEA) act as endogenous protective factors of the brain, using different pathways of neuroprotection against neuronal damage. Although several in vivo and in vitro studies confirmed the neuroprotective efficacy of endocannabinoids, no experimental settings compare and explore the neuroprotective potential of AEA and PEA in an acute stroke model. In this study, we investigated the neuroprotective potential by infarct measurement after high (30 mg/kg body weight) and low dosage administration (10 mg/kg body weight) of the endocannabinoid PEA in 49 male Wistar rats. In additions we studied infarct volumes of 22 male Wistar rats receiving the endocannabinoid AEA with a dosage of 10 mg/kg body weight or placebo. The neurological outcome was assessed 24 h after ischemia. Endocannabinoids were given intraperitoneally 30 min after initiation of transient middle cerebral artery occlusion (tMCAO). Infarct volume was calculated on the basis of 2.3.5-triphenyltetrazolium chloride staining. In the PEA high-dose group a significant total infarct reduction of 35% compared to the control group could be observed. AEA-treated rats presented a total infarct reducing effect of 26% compared to controls. Neurological scores, evaluated 24 h after tMCAO and physiological parameters, obtained 45 and 90 min after onset of ischemia showed no significant differences among the groups. As shown here, the endocannabinoids AEA and PEA achieved a significant neuroprotective effect by reducing size of infarcted tissue after tMCAO. Both endocannabinoids may have the potential to treat acute stroke and exert neuroprotection through a variety of mechanisms.

Autoreactive antibodies and loss of retinal ganglion cells in rats induced by immunization with ocular antigens.

PURPOSE In an experimental autoimmune animal model, retinal ganglion cell (RGC) loss was induced through immunization with glaucoma-related antigens. The target of this study was to investigate the pathomechanism behind this decline and the serum antibody reactivity against ocular and neuronal tissues after immunization with glaucoma- and non-glaucoma-associated antigens. METHODS Rats immunized with optic nerve antigen homogenate (ONA) or keratin (KER) were compared to control rats (CO). Intraocular pressure (IOP) was measured, and the fundi were examined regularly. Four weeks afterward, cells were counted in retinal flat mounts. Retina, optic nerve, and brain sections from healthy animals and optic nerve sections from immunized animals were incubated with serum collected at different time points. The occurrence of autoreactive antibodies was examined. Signs of antibody deposits, microglia activation, and demyelination were sought in optic nerves of immunized animals. Brain sections were examined for abnormalities. RESULTS No IOP or fundus changes were observed. Animals immunized with ONA showed a significant cell loss compared with the CO group. Elevated autoreactive antibodies against retina, optic nerve, and brain were observed. Animals immunized with KER, despite their immunologic response against KER, demonstrated neither RGC loss, nor increased development of autoreactive antibodies. Optic nerve from animals immunized with ONA demonstrated antibody accumulation, glia activation, and demyelination. No such observations were made in the KER or CO groups. Brain sections were without pathologic findings. CONCLUSIONS Systemic autoimmunity against ocular and neuronal epitopes, mediated by accordant autoreactive antibodies, is involved in the inflammatory processes that cause RGC degeneration in this experimental animal model.

Successful Regeneration After Experimental Stroke by Granulocyte-Colony Stimulating Factor Is Not Further Enhanced by Constraint-Induced Movement Therapy Either in Concurrent or in Sequential Combination Therapy

Background and Purpose— Both application of granulocyte-colony stimulating factor (G-CSF) and constraint-induced movement therapy (CIMT) have been shown to improve outcome after experimental stroke. The aim of the present study was to determine whether concurrent or sequential combination of both therapies will further enhance therapeutic benefit and whether specific modifications in the abundance of various neurotransmitter receptors do occur. Methods— Adult male Wistar rats were subjected to photothrombotic ischemia and assigned to the following treatment groups (n=20 each): (1) ischemic control (saline); (2) CIMT (CIMT between poststroke Days 2 and 11; (3) G-CSF (10 &mgr;g/kg G-CSF daily between poststroke Days 2 and 11; (4) combined concurrent group (CIMT plus 10 &mgr;g/kg G-CSF daily between poststroke Days 2 and 11; and (5) combined sequential group (CIMT between poststroke Days 2 and 11 and 10 &mgr;g/kg G-CSF daily between poststroke Days 12 and 21, respectively). Rats were functionally tested before and up to 4 weeks after ischemia. Quantitative receptor autography was performed for N-methyl-D-aspartate, AMPA, and GABAA receptors. Results— Significant improvement of functional outcome was seen in all groups treated with G-CSF alone and in either combination with CIMT, whereas CIMT alone failed to enhance recovery. Infarct sizes and remaining cortical tissue did not differ in the various treatment groups. Failure of significant benefit in the CIMT group was associated with a shift toward inhibition in perilesional and remote cortical regions. Conclusions— Our findings disclose G-CSF as the major player for enhanced recovery after experimental stroke, preventing a shift toward inhibition as seen in the CIMT group.

Eosinophilic Meningitis due to Angiostrongylus cantonensis in Germany

We report a case of eosinophilic meningitis due to Angiostrongylus cantonensis in a patient who returned from Thailand. The presence of a compatible epidemiologic history and eosinophilia in cerebrospinal fluid (CSF) lead to the diagnosis, which was confirmed by detection of specific antibodies. After treatment with albendazole and corticosteroids he recovered completely.

How much mutant protein is needed to cause a protein aggregate myopathy in vivo? Lessons from an exceptional desminopathy

Myofibrillar myopathies are caused by mutations in desmin, B‐crystallin, myotilin, ZASP, and filamin genes. Since the vast majority of myofibrillar myopathy causing mutations are heterozygous single amino acid substitutions or small in‐frame deletions, the pathogenic role of mutant versus wild‐type protein cannot be assessed in human skeletal muscle by standard immunodetection techniques. We report on an exceptional desminopathy due to a heterozygous c.735G>C mutation. Immunoblotting detected full‐length 53desmin and a truncated 50variant in skeletal muscle from three affected patients of two different families. RT‐PCR identified three desmin mRNA species encoding for wild‐type and two mutant proteins, p.Glu245Asp and p.Asp214_Glu245del. Since previous functional studies on the p.Glu245Asp mutant showed biological properties identical to wild‐type desmin, the truncated p.Asp214_Glu245del desmin is the disease‐causing mutant. Semiquantitative RT‐PCR established a fraction of the truncated desmin mRNA species in a range from 24% to 37%. Initial quantification of corresponding desmin proteins in the muscle biopsy of the index patient of one family indicated a fraction of only 10% of the truncated species. However, serial analyses of different sections from each muscle biopsy revealed a high intra‐ and interindividual variability of the truncated desmin protein level within a range from 5% to 43%. Desmin assembly studies in vitro have established clear‐cut pathogenic ratios of mutant versus wild‐type proteins. However, our findings point out a far more complex situation in human skeletal muscle. The heterogeneously distributed mutation load within and between individual specimens, which reflects local differences in the expression and/or turnover of the mutant protein in different areas containing multiple myonuclear domains, renders it impossible to define an exact pathogenic threshold of a specific mutant in vivo.

Does delta-sarcoglycan-associated autosomal-dominant cardiomyopathy exist?

In this study we clinically and genetically characterize a consanguineous family with a homozygous novel missense mutation in the δ-sarcoglycan gene and a second δ-sarcoglycan mutation that has previously been reported to cause severe autosomal-dominant dilated cardiomyopathy. We identified a novel missense mutation in exon 6 (p.A131P) of the δ-sarcoglycan gene, which in a homozygous state leads to the clinical picture of a limb girdle muscular dystrophy. In four heterozygous carriers for the mutation, aged 3–64 years, a second sequence variant in exon 6 (p.S151A) of the δ-sarcoglycan gene was detected on the other allele. This second missense change had previously been reported to be responsible for fatal autosomal-dominant dilated cardiomyopathy at young age. Comprehensive clinical and cardiac investigation in all of the compound heterozygous family members revealed no signs of cardiomyopathy or limb girdle muscular dystrophy. Our findings demonstrate that, even in the presence of a second disease-causing mutation, the p.S151A mutation in the δ-sarcoglycan gene does not result in cardiomyopathy. This finding questions the pathological relevance of this sequence variant for causing familial autosomal-dominant dilated cardiomyopathy and thereby the role of the δ-sarcoglycan gene in general as a disease-causing gene for autosomal-dominant dilated cardiomyopathy.

Waterjet dissection of peripheral nerves: an experimental study of the sciatic nerve of rats.

BACKGROUND: Although waterjet dissection has been well evaluated in intracranial pathologies, little is known of its qualities in peripheral nerve surgery. Theoretically, the precise dissection qualities could support the separation of nerves from adjacent tissues and improve the preservation of nerve integrity in peripheral nerve surgery. OBJECTIVE: To evaluate the potential of the new waterjet dissector in peripheral nerve surgery. METHODS: Waterjet dissection with pressures of 20 to 80 bar was applied on the sciatic nerves of 101 rats. The effect of waterjet dissection on the sciatic nerve was evaluated by clinical tests, neurophysiological examinations, and histopathological studies up to 12 weeks after surgery. RESULTS: With waterjet pressures up to 30 bar, the sciatic nerve was preserved in its integrity in all cases. Functional damaging was observed at pressures of 40 bar and higher. However, all but 1 rat in the 80 bar subgroup showed complete functional regeneration at 12 weeks after surgery. Histopathologically, small water bubbles were observed around the nerves. At 40 bar and higher, the sciatic nerves showed signs of direct nerve injury. However, all these animals showed nerve regeneration after 12 weeks, as demonstrated by histological studies. CONCLUSION: Sciatic nerves were preserved functionally and morphologically at pressures up to 30 bar. Between 40 and 80 bar, reliable functional and morphological nerve regeneration occurred. Waterjet pressures up to 30 bar might be applied safely under clinical conditions. This technique might be well suited to separate intact peripheral nerves from adjacent tumor or scar tissue. Further studies will have to show the clinical relevance of these dissection qualities.

Different postischemic protein expression of the GABA_{A} receptor α2 subunit and the plasticity-associated protein MAP1B after treatment with BDNF versus G-CSF in the rat brain

PURPOSE Recent data indicate that both brain-derived neurotrophic factor (BDNF) and granulocyte-colony stimulating factor (G-CSF) exert substantial neuroregenerative effects and improve functional outcome after ischemic stroke. In the present study, we checked for potential differences in the postischemic modulation of various excitatory and inhibitory neurotransmitter receptors as well as various marker molecules for structural plasticity by BDNF versus G-CSF. METHODS Adult male Wistar rats were subjected to photothrombotic ischemia and subsequently treated with NaCl, BDNF or G-CSF, respectively. After 6 weeks, postischemic protein expression of the NR1, GluR1 and alpha2 subunit of the NMDA, AMPA and GABA(A) receptor, respectively, was semiquantitatively determined ipsi- and contralateral to the ischemic lesion. Structural plasticity was further analyzed immunohistochemically using antibodies against MAP1B, MAP2 and synaptophysin. RESULTS Only BNDF caused a significantly reduced postischemic protein expression of the GABA(A) receptor alpha2 subunit and the NR1 subunit of the NMDA receptor in the hippocampus. Furthermore, BDNF compared to G-CSF increased MAP1B protein expression in the periischemic regenerative region. CONCLUSIONS Although both BDNF and G-CSF have been shown to improve postischemic functional outcome to a similar extent, exogenous administration results in different underlying structural reorganization processes suggesting specific modulations of plasticity-associated events by these trophic factors.