Dorota Sulejczak

Neuroprotective effects of tempol on retinal ganglion cells in a partial optic nerve crush rat model with and without iron load.

Iron overload can contribute to oxidative stress in many tissues. We studied the effects of pretreatment with iron dextran on RGC loss in a calibrated partial optic nerve crush (PONC) model in rats, along with the protection offered by tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl, a membrane-permeable superoxide dismutase mimetic and free-radical scavenger), in the same experimental paradigm. A total of 40 rats in 6 groups of 5-8 animals each underwent PONC in one eye and sham crush in the other. Animals were pretreated with a single iron dextran load 24 h prior to PONC, and treated with tempol 6 h before and then once daily after PONC. Control animals were treated with PBS. RGC were retrogradely labeled with a fluorescent marker; all data are expressed in percent of the RGC count in the respective sham-treated eye. Immunohistochemistry was performed to visualize 3-nitrotyrosine, a marker of nitroxidative stress. PONC without iron pretreatment resulted in the survival of only 31.4% of labeled RGC after 7 days. Even fewer RGC (12.7%) survived after PONC with iron pretreatment. However, tempol in doses of 20 mg/kg of body weight (BW) significantly attenuated this effect when given as described above; in the group without iron pretreatment the number of surviving RGC doubled from 31.4% to 62.1%. In the group with iron pretreatment the survival rate of RGC increased even more pronouncedly, from 12.7% without tempol to 46.2% with tempol. Tempol in doses of 1 mg/kg BW and 5 mg/kg BW showed no significant rescue of RGC. Immunostaining showed nitrotyrosine-positive RGCs in PONC but not in sham-treated eyes and an increase in positive cells after iron load. Tempol treatment reduced nitrotyrosine staining in both the iron and non-iron groups. Our results demonstrate that PONC results in significantly greater RGC damage when iron pretreatment is performed, and that the compound tempol may provide additional protection for RGC in cases of neuronal damage both with and without prior iron treatment.

Temporal expression of P2X7 purinergic receptor during the course of experimental autoimmune encephalomyelitis.

Purinergic P2X(7) receptors are nucleotide-gated ion channels widely distributed in brain. Strong evidence suggests that they are involved in cross-talk between glial and neuronal cells. These receptors activated under pathological conditions may participate in regulation of inflammatory response and cell death. In this study we show the expression of P2X(7) protein and mRNA during the course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), in different stages of the disease (4, 6, 8, 10 post-immunization). The enhanced expression of the receptor at the level of both mRNA and protein was observed in the peak of neurological symptoms and was connected mostly with neurons. However, early overexpression of receptor protein was observed also in an asymptomatic phase of EAE and was tightly related to astrocytic pool of cells. This suggests the early involvement of this kind of receptor into pathological mechanisms leading for symptoms characteristic for EAE.

Neurofilament heavy chain and heat shock protein 70 as markers of seizure-related brain injury.

Purpose:  Status epilepticus (SE) has deleterious effects on brain tissue, but whether brief recurrent seizures may also damage neurons represents a matter of controversy. Therefore, it remains a central area of epilepsy research to identify individuals at risk where disease progression can be potentially prevented. Biomarkers may serve as tools for such identification. Thus the present study aimed at analyzing the levels of heat shock protein 70 (HSP‐70, also designated as HSPA1A) and neurofilament heavy chain protein (NfHSMI35) in cerebrospinal fluid (CSF) of patients with seizures of different severity.

Diverse Expression of Selected SMN Complex Proteins in Humans with Sporadic Amyotrophic Lateral Sclerosis and in a Transgenic Rat Model of Familial Form of the Disease

Background and Objective There is circumstantial evidence linking sporadic amyotrophic lateral sclerosis (ALS) cases to a malfunction or deficit of a multimeric SMN complex that scrutinizes cellular RNAs; the core of this complex is survival motor neuron (SMN, or gemin 1) protein. We intended to verify this hypothesis by comparing the expression of both SMN and several other functionally associated gemins in the anterior horn motoneurons of patients who died of sporadic ALS (sALS), of transgenic rats with overexpression of the mutated human superoxide dismutase 1 gene (SOD1G93A) that represent a model of familial ALS (fALS), and of the respective controls. Methods Using archival material of paraffin blocks with samples of human and rat spinal cords, immunohistochemical reactions with antibodies against SMN and gemins 2, 3, and 4 were performed and assessed by light microscopy. Results The expression of SMN and all other studied gemins was observed in motoneurons of sALS patients, fALS rats, and in all controls, although the intensity varied. The immunolabeling was most intense in sALS patients with relatively fast disease course, and decreased with increasing disease duration in both the human sALS and rat fALS material. Irrespective of the disease stage, sALS material showed no or very low gemin 2 immunoreactivity, while clear gemin 2 immnoreactivity was observed in all fALS rats and control material. Conclusion The deficient expression of gemin 2 in spinal cord motoneurons in human sALS may lead to a dysfunction and loss of neuroprotective action of the SMN complex.

Anandamide enhances expression of heat shock proteins Hsp70 and Hsp25 in rat lungs

Anandamide (AEA), an endogenous cannabinoid and vanilloid receptor ligand, possesses anti-inflammatory properties. Transport of AEA through cytoplasm is facilitated by heat shock protein (HSP) Hsp70, which enhances the rate of cellular AEA uptake, possibly via direct interactions. In lungs, increased HSP expression is an endogenous, protective mechanism against acute lung inflammation. We hypothesised that AEA could enhance the expression of cytoprotective Hsp70 and Hsp25. Anaesthetised rats were injected intravenously with 1mg/kg AEA or saline. Lungs were removed 2 and 24 h after injection for evaluation of HSP expression. Hsp70 and Hsp25 expression in lungs was evaluated by immunohistochemistry. The relative levels of these HSPs in lung sections were determined through optical density measurements and western blotting of lung homogenates. Western blot and immunohistochemistry analyses indicated that expression of both proteins was significantly higher in AEA-injected animals than in control animals 2 and 24 h after treatment. AEA administration enhanced Hsp70 and Hsp25 expression in lungs. Therefore, AEA-HSP interactions could be involved in mechanisms protecting against lung inflammation, indicating a possible use of AEA as a treatment for lung inflammation.

The role of allogenic keratin - derived dressing in wound healing in a mouse model.

Keratin is an interesting protein needed for wound healing and tissue recovery. We have recently proposed a new, simple and inexpensive method to obtain fur and hair keratin‐derived biomaterials suitable for medical application. The aim of the study was to evaluate the role of the fur keratin‐derived protein (FKDP) dressing in the allogenic full‐thickness surgical skin wound model. The data obtained using scanning electron microscopy showed that employed processed biomaterial had higher surface porosity compared with control raw material. From the MTS test, it was found keratin biomaterial is not only toxic to the NIH/3T3 cell line (p < 0.05), but also enhances cell proliferation compared with the control. In vivo studies have shown keratin dressings are tissue biocompatible, accelerate wound closure and epithelialization to the statistically significant differences on day 5 (p < 0.05) in comparison to control wounds. Histological examination revealed, that in FKDP‐treated wounds the inflammatory response contained predominantly macrophages whilst their morphological untreated variants showed mixed cell infiltrates rich in neutrophils. Predominant macrophages based response creates more favorable environment for healing. In FKDP‐dressed wounds the number of microhemorrhages was also significantly decreased (p < 0.05) as compared with undressed wounds. Applied keratin dressing favors reconstruction of a more regular skin structure and assures better cosmetic effect in terms of scar formation and appearance. In conclusion, fur keratin‐derived protein dressings significantly accelerated wound healing in the mouse model. Further studies are needed to determine the molecular mechanisms involved in the multilayer wound healing process and to assess the possible use of these dressings for medical purposes.

Ultrastructural and Immunochemical Studies of Glial Scar Formation in Diabetic Rats

We explored the rebuilding of the brain parenchyma after surgical injury due to reactive astrogliosis. In the present study, we investigated the initial stages of rebuilding in the perilesional cortex of streptozotocin-induced diabetic rats. Our methods utilized ultrastructural and immunohistochemical studies as well as Western blot analysis of glial fibrillary acidic protein (GFAP) and vimentin. Data was collected at 2 days, 7 days and 2 months following a unilateral sensorimotor cortex lesion. Electron-microscopic studies revealed not only formation of glial scar tissue but also ultrastructural features of death in the elements of neurovascular unit. Immunohistochemical studies, confirmed by Western blot analysis, demonstrated the enhancement of vimentin and GFAP immunoreactivity (IR) in astrocytes located in the perilesion cortical area of the diabetic rats that were operated upon. We suggest that the process of rebuilding brain parenchyma following surgical injury may be disturbed by the induction of astrocytes and the degeneration of astrocytes, as well as by morphological changes within capillaries that are accompanied by the presence of macrophages.

What factors determine phenotype of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)? Considerations in the context of a novel pathogenic R110C mutation in the NOTCH3 gene

We report patients from a Polish family with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) who possess a novel heterozygous R110C mutation in exon 3 of the NOTCH3 gene leading to stereotypical cysteine loss. The proband had only seizure attacks and her magnetic resonance imaging (MRI) showed very numerous hyperintense foci in the cerebral white matter in a location characteristic of CADASIL. Distinctive ultrastructural assessment of vessels from skin-muscle biopsy revealed only mild degenerative changes but relatively numerous homogeneous deposits of granular osmiophilic material (GOM). In the other symptomatic family members with the same mutation ischaemic strokes were present but not epilepsy. In the probands affected brother at a similar age, the brain MRI was normal but vessels showed pronounced degenerative changes and irregular GOM deposits. The present report not only extends the list of known pathogenic mutations responsible for CADASIL but also emphasizes clinical and morphologic variability among family members with the same NOTCH3 mutation, suggesting that probably additional factors, not only mutations, may influence the disease phenotype..