Oliver Herrmann

IKK mediates ischemia-induced neuronal death

The IκB kinase complex IKK is a central component of the signaling cascade that controls NF-κB–dependent gene transcription. So far, its function in the brain is largely unknown. Here, we show that IKK is activated in a mouse model of stroke. To investigate the function of IKK in brain ischemia we generated mice that contain a targeted deletion of Ikbkb (which encodes IKK2) in mouse neurons and mice that express a dominant inhibitor of IKK in neurons. In both lines, inhibition of IKK activity markedly reduced infarct size. In contrast, constitutive activation of IKK2 enlarged the infarct size. A selective small-molecule inhibitor of IKK mimicked the effect of genetic IKK inhibition in neurons, reducing the infarct volume and cell death in a therapeutic time window of 4.5 h. These data indicate a key function of IKK in ischemic brain damage and suggest a potential role for IKK inhibitors in stroke therapy.

Neuronal Activation of NF-κB Contributes to Cell Death in Cerebral Ischemia

The transcription factor NF-κB is a key regulator of inflammation and cell survival. NF-κB is activated by cerebral ischemia in neurons and glia, but its function is controversial. To inhibit NF-κB selectively in neurons and glial cells, we have generated transgenic mice that express the IκBα superrepressor (IκBα mutated at serine-32 and serine-36, IκBα-SR) under transcriptional control of the neuron-specific enolase (NSE) and the glial fibrillary acidic protein (GFAP) promoter, respectively. In primary cortical neurons of NSE-IκBα-SR mice, NF-κB activity was partially inhibited. To assess NF-κB activity in vivo after permanent middle cerebral artery occlusion (MCAO), we measured the expression of NF-κB target genes by real-time polymerase chain reaction (PCR). The induction of c-myc and transforming growth factor-β2 by cerebral ischemia was inhibited by neuronal expression of IκBα-SR, whereas induction of GFAP by MCAO was reduced by astrocytic expression of IκBα-SR. Neuronal, but not astrocytic, expression of the NF-κB inhibitor reduced both infarct size and cell death 48 hours after permanent MCAO. In summary, the data show that NF-κB is activated in neurons and astrocytes during cerebral ischemia and that NF-κB activation in neurons contributes to the ischemic damage.

Regulation of body temperature and neuroprotection by endogenous interleukin-6 in cerebral ischemia.

Although the function of fever is still unclear, it is now beyond doubt that body temperature influences the outcome of brain damage. An elevated body temperature is often found in stroke patients and denotes a bad prognosis. However, the pathophysiologic basis and treatment options of elevated body temperature after stroke are still unknown. Cerebral ischemia rapidly induced neuronal interleukin-6 (IL-6) expression in mice. In IL-6–deficient mice, body temperature was markedly decreased after middle cerebral artery occlusion (MCAO), but infarct size was comparable to that in control mice. If body temperature was controlled by external warming after MCAO, IL-6–deficient mice had a reduced survival, worse neurologic status, and larger infarcts than control animals. In cell culture, IL-6 exerted an antiapoptotic and neuroprotective effect. These data suggest that IL-6 is a key regulator of body temperature and an endogenous neuroprotectant in cerebral ischemia. Neuroprotective properties apparently compensate for its pyretic action after MCAO and enhance the safety of this endogenous pyrogen.

Bim and Noxa Are Candidates to Mediate the Deleterious Effect of the NF-κB Subunit RelA in Cerebral Ischemia

The transcription factor nuclear factor κB (NF-κB) is well known for its antiapoptotic action. However, in some disorders, such as cerebral ischemia, a proapoptotic function of NF-κB has been demonstrated. To analyze which subunit of NF-κB is functional in cerebral ischemia, we induced focal cerebral ischemia in mice with a germline deletion of the p52 or c-Rel gene or with a conditional deletion of RelA in the brain. Only RelA deficiency reduced infarct size. Interestingly, expression of the proapoptotic BH3 (Bcl-2 homology domain 3)-only genes Bim and Noxa in cerebral ischemia depended on RelA and the upstream kinase IKK (IκB kinase). RelA stimulated Bim and Noxa gene transcription in primary cortical neurons and bound to the promoter of both genes. Thus, the deleterious function in cerebral ischemia is specific for the NF-κB subunit RelA and may be mediated through Bim and Noxa.

Functional testing in a mouse stroke model induced by occlusion of the distal middle cerebral artery

Reducing post-stroke disability is the major goal of stroke therapy. Consequently, functional testing is essential in experimental stroke studies to increase the predictive value of animal models. We used several sensory and motor tests to assess functional disability in a mouse model of permanent distal middle cerebral artery occlusion (pdMCAO) that induced mainly cortical infarcts. Gait dynamics were transiently disturbed after pdMCAO as measured by different analysis techniques. Stance and brake duration were shorter after pdMCAO. Consistent with sensory and motor deficits the latency to move was prolonged up to 14 days after pdMCAO and the performance in the corner test and handedness were affected on day 1 or 2 after pdMCAO. Heart rate was decreased and heart rate variability were increased after pdMCAO indicating sympathetic-parasympathetic imbalance. In summary, pdMCAO-induced cortical infarcts lead to clinically relevant sensory, motor and cardiac autonomic dysfunction in mice. The present study provides a basis to explore the potential of functional testing for neuroprotection and neuroregeneration after stroke.

NF-κB signalling in cerebral ischaemia

In acute stroke, neuronal apoptosis and inflammation are considered to be important mechanisms on the road to tissue loss and neurological deficit. Both apoptosis and inflammation depend on gene transcription. We have identified a signalling pathway that regulates transcription of genes involved in apoptosis and inflammation. In a mouse model of focal cerebral ischaemia, there is an induction of the cytokine TWEAK (tumour necrosis factor-like weak inducer of apoptosis) and its membrane receptor Fn14. TWEAK promotes neuronal cell death and activates the transcription factor NF-kappaB (nuclear factor kappaB) through the upstream kinase IKK [IkappaB (inhibitory kappaB) kinase]. In vivo, IKK is activated in neurons. Neuron-specific deletion of the subunit IKK2 or inhibition of IKK activity reduced the infarct size and neuronal cell loss. A pharmacological inhibitor of IKK also showed neuroprotective properties. IKK-dependent ischaemic brain damage is likely to be mediated by NF-kappaB, because neuron-specific inhibition of NF-kappaB through transgenic expression of the NF-kappaB superrepressor was found to reduce the infarct size. In summary, there is evidence that IKK/NF-kappaB signalling contributes to ischaemic brain damage and may provide suitable drug targets for the treatment of stroke.

Oxygen therapy in permanent brain ischemia: Potential and limitations

BACKGROUND Both normobaric (NBO) and hyperbaric (HBO) oxygen therapy are protective in transient cerebral ischemia. In contrast, in permanent ischemia models, which reflect the majority of clinical strokes, the effectiveness of NBO is unknown, and the effectiveness of HBO is controversial. The goals of the present study were to compare both oxygen therapies in 2 models of permanent ischemia, to study the effect of time window, and to evaluate the combination of both oxygen therapies. METHODS Distal or proximal permanent occlusion of middle cerebral artery (MCAO) was induced by coagulation or filament, respectively. Mice received air, NBO, a single or repeated HBO (3 ata) treatments. Infarct sizes were quantified at 7 days (coagulation) and 24 h (filament), respectively. RESULTS Following MCA coagulation, infarct volume was 12.9+/-1.6 mm3 in mice breathing air. When started 45 min or 120 min after MCAO, NBO (10.8+/-2.2) and significantly more potently HBO (7.8+/-0.9) reduced infarct size. Repeated HBO treatments had no additional effect (8.3+/-2.3). HBO also significantly decreased TUNEL cell staining at 24 h. Combination of 60 min NBO plus 60 min HBO resulted in smaller cortical infarcts (8.7+/-1.5) than 120 min NBO alone (11.1+/-3.2). In contrast, infarct volumes in filament-induced permanent MCAO did not differ among rodents receiving air (50+/-24 mm3), NBO (48+/-16), or HBO (46+/-21). After filament-induced transient MCAO, however, HBO reduced infarct volume significantly. CONCLUSIONS NBO and more effectively HBO protect the brain against permanent cortical ischemia. In extensive focal ischemia, however, oxygen therapy is only effective in case of early recanalization.

Blood Pressure Variability after Intravenous Thrombolysis in Acute Stroke Does Not Predict Intracerebral Hemorrhage but Poor Outcome

Background: The relevance of blood pressure variability (BPV) in the development of intracerebral hemorrhage (ICH) after intravenous thrombolysis (IVT) in acute stroke still remains uncertain. Methods: 427 consecutive patients treated with IVT in the years 2007–2009 were studied. Blood pressure (BP) values were analyzed from admission to follow-up imaging scan and described as mean, maximum, minimum, standard deviation (SD), difference between maximum and minimum, successive variation (SV) and maximum SV. ICH was categorized based on radiologic criteria and symptomatic ICH (sICH) was defined as ICH plus worsening of the National Institute of Health Stroke Scale by ≧4 points or leading to death. Three-month outcome was described by means of the modified Rankin Scale. Results: We observed any ICH in 51 (11.9%) and sICH in 10 (2.3%) patients. Systolic and diastolic BP profiles, including mean, maximum, minimum, SD, difference between maximum and minimum, SV and maximum SV, did not differ between ICH-negative, ICH-positive and sICH patients. In univariate analysis, high systolic BPV was associated with sICH (p = 0.03). A logistic regression model to predict ICH only found early CT findings (OR = 2.74, 95% CI = 1.47–5.11, p < 0.01) as independently associated with ICH. Poor 3-month outcome was independently predicted by age (OR = 0.96, 95% CI = 0.94–0.97, p < 0.001), NIHSS on admission (OR = 0.84, 95% CI = 0.80–0.87, p < 0.001), ICH (OR = 0.29, 95% CI = 0.13–0.66, p < 0.01) and high systolic BPV (OR = 1.68, 95% CI = 1.05–2.69, p < 0.05). Conclusions: We demonstrate that high BPV in patients receiving IVT leads to poor outcome but does not increase the risk of ICH/sICH.

Clinical and genetic variability of oculodentodigital dysplasia.

To the Editor: Oculodentodigital dysplasia (ODDD, OMIM 164200) is a rare autosomal dominant inherited disorder affecting the development of face, eyes, teeth, and limbs. A key finding is syndactyly III affecting fingers V and IV, sometime also the fingers IV and III and toes. Other typical signs include microphthalmos, a small nose, hypotrichosis, and abnormal dentition. In addition, several patients develop neurological problems in adulthood, mostly a spastic paraparesis associated with white matter hyperintensity onMR imaging (1). The syndrome is caused by mutations of the gene GJA1 encoding for connexin-43 (2–4). In most cases, missense mutations in the 50-end of the GJA1 gene were found. The phenotypes range from syndactyly III alone to ODDD without syndactyly (3, 4). Although neurological symptoms are underdiagnosed due to the late occurrence in the course of the disease, it is clear that some families are not affected by neurological symptoms (2). Here, we describe four GJA1 mutations in ODDD patients, two of which were associated with a spastic paraparesis and one with a developmental and language disorder. Families 1 and 2 were described previously (1, 5). However, in family 1, no attention was paid to neurological symptoms (5). Patient 1-1 had the first symptoms in her thirties (Table 1). T2weightedMRI of the brain disclosed a hyperintensive signal in the white matter adjacent to the posterior horns. Visual-, auditory-, and somatosensory-evokedpotentialswere delayed. Patient 1-2 (daughter of 1-1) suffered from occasional faecal incontinence at the age of 15, but there were no other neurological symptoms. Visualand auditory-evoked potentials were abnormal. In patient 4-1, attention deficit and impaired language developmentwere observed at the age of 3. For genotyping, the coding sequence of the GJA1 gene was amplified and sequenced as describedbefore (2). In 20 healthy subjects, in three unaffected sisters of patient 1-1, and in the parents of patients 3-1 and 4-1, none of the described mutations was found. ODDD is a pleiotropic syndrome involving several tissues such as the white matter of the nervous system, in which connexin-43 is forming gap junctions. Heterozygote mutations of one GJA1 allele are generally thought to exert a dominant negative effect on the function of multisubunit gap junctions (6, 7). This explains the dominant inheritance of the disorder. The function and interaction partners of connexin-43 vary with the tissue (8). Thus, a specific mutation may affect gap junctions in only a subset of connexin-43 expressing tissues. Indeed, the presentation of ODDD differs between families and possibly depends on the specific connexin-43 mutation. In our teenage patient 3-1 and in a Danish family, missense mutations of codon 96 were not associated with a neurological phenotype although our patient may still be too young to manifest neurological symptoms (9). Neurological signs were also missing with other mutations (S18P, A40V, Q49K, F52dup, Y98C, E110D, and R202H) (2, 10). However, we have detected three mutations in patients with neurological symptoms (spastic paraparesis in patients 1-1, 2-1; developmental disorder in patient 4-1), adding to a number of previously described missense mutations linked to neurological signs (Y17S, G22E, K23T, L90V, K102N, I130T, K134E, G138R, V216L, K134N, and R148Q) (2, 3). Thus, mutations associated with neurological symptoms are spread over the N-terminal part of connexin-43 and cannot be localized to a certain domain of the protein. Expression of ODDD-associated connexin-43 mutations in a glial cell line resulted in non-functional connexin hemichannels and gap junction functions regardless of whether the particular mutant is associated with neurological dysfunction (11). Thus, up to now it is unclear which factors determine whether a mutation leads to neurological symptoms or not. Most mutations are specific for a family. Of the four mutations we have detected, only L113P (T338C) has been described before (3). In addition, previous reports and two of our patients (3-1, 4-1) demonstrate that new mutations occur in the gene.

Restriction-mediated differential display (RMDD) identifies pip92 as a pro-apoptotic gene product induced during focal cerebral ischemia

Studies of gene expression changes after cerebral ischemia can provide novel insight into ischemic pathophysiology. Here we describe application of restriction-mediated differential display to screening for differentially expressed genes after focal cerebral ischemia. This method combines the nonredundant generation of biotin-labeled fragment sets with the excellent resolution of direct blotting electrophoresis, reliable fragment recovery, and a novel clone selection strategy. Using the filament model in mouse with 90 minutes MCA occlusion followed by 2, 6, and 20 hours reperfusion, we have compared gene expression in sham-operated animals to both the ipsi- and contralateral forebrain hemisphere of ischemic mice. Our screening method has resulted in the identification of 70 genes differentially regulated after transient middle cerebral artery occlusion (MCAO), several of which represent unknown clones. We have identified many of the previously published regulated genes, lending high credibility to our method. Surprisingly, we detected a high degree of correspondent regulation of genes in the nonischemic hemisphere. A high percentage of genes coding for proteins in the respiratory chain was found to be up-regulated after ischemia, potentially representing a new mechanism involved in counteracting energy failure or radical generation in cerebral ischemia. One particularly interesting gene, whose upregulation by ischemia has not been described before, is pip92; this gene shows a rapid and long-lasting induction after cerebral ischemia. Here we demonstrate that pip92 induces cell death in primary neurons and displays several hallmarks of pro-apoptotic activity upon overexpression, supporting the notion that we have identified a novel pathophysiological player in cerebral ischemia. In summary, restriction-mediated differential display has proven its suitability for screening complex samples such as brain to reliably identify regulated genes, which can uncover novel pathophysiological mechanisms.