Anis Khusro Mir

Nogo-A Antibody Improves Regeneration and Locomotion of Spinal Cord-Injured Rats

Spinal cord trauma leads to loss of motor, sensory and autonomic functions below the lesion. Recovery is very restricted, due in part to neurite growth inhibitory myelin proteins, in particular Nogo‐A. Two neutralizing antibodies against Nogo‐A were used to study recovery and axonal regeneration after spinal cord lesions. Three months old Lewis rats were tested in sensory‐motor tasks (open field locomotion, crossing of ladder rungs and narrow beams, the CatWalk® runway, reactions to heat and von Frey hairs). A T‐shaped lesion was made at T8, and an intrathecal catheter delivered highly purified anti‐Nogo‐A monoclonal IgGs or unspecific IgGs for 2 weeks. A better outcome in motor behavior was obtained as early as two weeks after lesion in the animals receiving the Nogo‐A antibodies. Withdrawal responses to heat and mechanical stimuli were not different between the groups. Histology showed enhanced regeneration of corticospinal axons in the anti‐Nogo‐A antibody groups. fMRI revealed significant cortical responses to stimulation of the hindpaw exclusively in anti‐Nogo‐A animals. These results demonstrate that neutralization of the neurite growth inhibitor Nogo‐A by intrathecal antibodies leads to enhanced regeneration and reorganization of the injured CNS, resulting in improved recovery of compromised functions in the absence of dysfunctions. Ann Neurol 2005

Nogo-A–specific antibody treatment enhances sprouting and functional recovery after cervical lesion in adult primates

In rodents, after spinal lesion, neutralizing the neurite growth inhibitor Nogo-A promotes axonal sprouting and functional recovery. To evaluate this treatment in primates, 12 monkeys were subjected to cervical lesion. Recovery of manual dexterity and sprouting of corticospinal axons were enhanced in monkeys treated with Nogo-A–specific antibody as compared to monkeys treated with control antibody. NOTE: In the version of this article initially published, the cut corticospinal tract (CST) stumps rostral to the lesion site in Figure 2d and Supplementary Fig. 3a online were meant to be represented schematically, a fact not explained in the figure legend. These representations should therefore have been replaced by full camera lucida reconstructions of these rostral cut CST stumps for the corresponding animals, requiring the consideration of additional sections of the spinal cord located more laterally than those drawn here for the reconstruction of the CST axonal arbors caudal to the lesion (sections for which the contours are represented here). The figure has been corrected in the HTML and the PDF versions of the article.

Monocyte chemoattractant protein-1 deficiency is protective in a murine stroke model

Inflammatory processes have been implicated in the pathogenesis of brain damage after stroke. In rodent stroke models, focal ischemia induces several proinflammatory chemokines, including monocyte chemoattractant protein-1 (MCP-1). The individual contribution to ischemic tissue damage, however, is largely unknown. To address this question, the authors subjected MCP-1-deficient mice (MCP-1−/−) to permanent middle cerebral artery occlusion (MCAO). Measurement of basal blood pressure, cerebral blood flow, and blood volume revealed no differences between wild-type (wt) and MCP-1−/− mice. MCAO led to similar cerebral perfusion deficits in wt and MCP-1−/− mice, excluding differences in the MCA supply territory and collaterals. However, compared with wt mice, the mean infarct volume was 29% smaller in MCP-1−/− mice 24 hours after MCAO (P = 0.022). Immunostaining showed a reduction of phagocytic macrophage accumulation within infarcts and the infarct border in MCP-1−/− mice 2 weeks after MCAO. At the same time point, the authors found an attenuation of astrocytic hypertrophy in the infarct border and thalamus in MCP-1−/− mice. However, these effects on macrophages and astrocytes in MCP-1−/− mice occurred too late to suggest a protective role in acute infarct growth. Of note: at 6 hours after MCAO, MCP-1−/− mice produced significantly less interleukin-1β in ischemic tissue; this might be related to tissue protection. The results of this study indicate that inhibition of MCP-1 signaling could be a new acute treatment approach to limit infarct size after stroke.

Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, during acute and chronic inflammation in the rodent CNS

In this study, we investigate the expression of fractalkine (CX3CL1) and the fractalkine receptor (CX3CR1) in the naive rat and mouse central nervous system (CNS). We determine if the expression of this chemokine and its receptor are altered during chronic or acute inflammation in the CNS. In addition, we determine if CX3CL1, which has been reported to be chemoattractant to leukocytes in vitro, is capable of acting as a chemoattractant in the CNS in vivo. Immunohistochemistry was performed using primary antibodies recognizing soluble and membrane‐bound CX3CL1 and the N‐terminus of the CX3CR1. We found that neurons in the naive rodent brain are immunoreactive for CX3CL1 and CX3CR1, both showing a perinuclear staining pattern. Resident microglia associated with the parenchyma and macrophages in the meninges and choroid plexus constituitively express CX3CR1. In a prion model of chronic neurodegeneration and inflammation, CX3CL1 immunoreactivity is upregulated in astrocytes and CX3CR1 expression is elevated on microglia. In surviving neurons, expression of CX3CL1 appears unaltered relative to normal neurons. There is a decrease in neuronal CX3CR1 expression. Acute inflammatory responses in the CNS, induced by stereotaxic injections of lipopolysaccharide or kainic acid, results in activation of microglia and astrocytes but no detectable changes in the glial expression of CX3CL1 or CX3CR1. The expression of CX3CL1 and CX3CR1 by glial cells during inflammation in the CNS may be influenced by the surrounding cytokine milieu, which has been shown to differ in acute and chronic neuroinflammation. GLIA 37:314–327, 2002.

Anti–Nogo-A Antibody Infusion 24 Hours After Experimental Stroke Improved Behavioral Outcome and Corticospinal Plasticity in Normotensive and Spontaneously Hypertensive Rats

Nogo-A is a myelin-associated neurite outgrowth inhibitory protein limiting recovery and plasticity after central nervous system injury. In this study, a purified monoclonal anti—Nogo-A antibody (7B12) was evaluated in two rat stroke models with a time-to-treatment of 24 hours after injury. After photothrombotic cortical injury (PCI) and intraventricular infusion of a control mouse immunoglobulin G for 2 weeks, long-term contralateral forepaw function was reduced to about 55% of prelesion performance until the latest time point investigated (9 weeks). Forepaw function was significantly better in the 7B12-treated group 6 to 9 weeks after PCI, and reached about 70% of prelesion levels. Cortical infarcts were also produced in spontaneously hypertensive rats (SHR) by permanent middle cerebral artery occlusion (MCAO). In the control group, forepaw function remained between 40% and 50% of prelesion levels 4 to 12 weeks after MCAO. In contrast, 7B12-treated groups showed significant improvement between 4 and 7 weeks after MCAO from around 40% of prelesion levels at week 4 to about 60% to 70% at 7 to 12 weeks after MCAO. Treatment in both models was efficacious without influencing infarct volume or brain atrophy. Neuroanatomically in the spinal cord, a significant increase of midline crossing corticospinal fibers originating in the unlesioned sensorimotor cortex was found in 7B12-treated groups, reaching 2.3 ± 1.5% after PCI (control group: 1.1 ± 0.5%) and 4.5 ± 2.2% after MCAO in SHR rats (control group: 1.8 ± 0.8%). Behavioral outcome and the presence of midline crossing fibers in the cervical spinal cord correlated significantly, suggesting a possible contribution of the crossing fibers for forepaw function after PCI and MCAO. The results suggest that specific anti—Nogo-A antibodies bear potential as a new rehabilitative treatment approach for ischemic stroke with a prolonged time-to-treatment window.

Targeting monocyte chemoattractant protein-1 signalling in disease.

Monocyte chemoattractant protein-1 (MCP-1) has been implicated in many inflammatory and autoimmune diseases. The G-protein-coupled receptor CCR-2B is probably the most important MCP-1 receptor in vivo, and loss of MCP-1 effector function alone is sufficient to impair monocytic trafficking in inflammation models. MCP-1 signalling appears to be a relevant target, especially in rheumatoid arthritis (RA). In RA patients, MCP-1 is produced by synovial cells and infiltrating monocytes, plasma MCP-1 concentrations correlate with swollen joint count, and elevated serum MCP-1 concentrations were found in juvenile RA in patients with active disease. Modulation of MCP-1 signalling in experimental RA showed beneficial effects on inflammation and joint destruction. With respect to chronic neuroinflammation, a critical role for MCP-1 has been established in animal models for multiple sclerosis. In acute neuroinflammation, experimental evidence for a detrimental role of MCP-1 in stroke and excitotoxic injury has been found. Several selective small molecular weight CCR-2B antagonists and MCP-1-blocking antibodies have been described. The proof for the validity of targeting MCP-1 signalling in disease, however, has yet to be established in clinical trials.

Neutralization of interleukin-1β modifies the inflammatory response and improves histological and cognitive outcome following traumatic brain injury in mice

Interleukin‐1β (IL‐1β) may play a central role in the inflammatory response following traumatic brain injury (TBI). We subjected 91 mice to controlled cortical impact (CCI) brain injury or sham injury. Beginning 5 min post‐injury, the IL‐1β neutralizing antibody IgG2a/k (1.5 μg/mL) or control antibody was infused at a rate of 0.25 μL/h into the contralateral ventricle for up to 14 days using osmotic minipumps. Neutrophil and T‐cell infiltration and microglial activation was evaluated at days 1–7 post‐injury. Cognition was assessed using Morris water maze, and motor function using rotarod and cylinder tests. Lesion volume and hemispheric tissue loss were evaluated at 18 days post‐injury. Using this treatment strategy, cortical and hippocampal tissue levels of IgG2a/k reached 50 ng/mL, sufficient to effectively inhibit IL‐1βin vitro. IL‐1β neutralization attenuated the CCI‐induced cortical and hippocampal microglial activation (P < 0.05 at post‐injury days 3 and 7), and cortical infiltration of neutrophils (P < 0.05 at post‐injury day 7). There was only a minimal cortical infiltration of activated T‐cells, attenuated by IL‐1β neutralization (P < 0.05 at post‐injury day 7). CCI induced a significant deficit in neurological motor and cognitive function, and caused a loss of hemispheric tissue (P < 0.05). In brain‐injured animals, IL‐1β neutralizing treatment resulted in reduced lesion volume, hemispheric tissue loss and attenuated cognitive deficits (P < 0.05) without influencing neurological motor function. Our results indicate that IL‐1β is a central component in the post‐injury inflammatory response that, in view of the observed positive neuroprotective and cognitive effects, may be a suitable pharmacological target for the treatment of TBI.

Lipopolysaccharide induces expression of tumour necrosis factor alpha in rat brain: inhibition by methylprednisolone and by rolipram

1 We have investigated the effects of the phosphodiesterase (PDE) type IV inhibitor rolipram and of the glucocorticoid methylprednisolone on the induction of tumour necrosis factor alpha (TNF‐α) mRNA and protein in brains of rats after peripheral administration of lipopolysaccharide (LPS). 2 After intravenous administration of LPS, a similar time‐dependent induction of both TNF‐α mRNA and protein was observed in rat brain. Peak mRNA and protein levels were found 7 h after administration of LPS. 3 In situ hybridization experiments with a specific antisense TNF‐α riboprobe suggested that the cells responsible for TNF‐α production in the brain were microglia. 4 Intraperitoneal administration of methylprednisolone inhibited the induction of TNF‐α protein in a dose‐dependent manner. A maximal inhibition of TNF‐α protein production by 42.9±10.2% was observed at a dose regimen consisting of two injections of each 30 mg kg−1 methylprednisolone. 5 Intraperitoneal administration of rolipram also inhibited the induction of TNF‐α protein in a dose‐dependent manner. The maximal inhibition of TNF‐α protein production was 96.1±12.2% and was observed at a dose regimen of three separate injections of each 3 mg kg−1 rolipram. 6 In situ hybridization experiments showed that the level of TNF‐α mRNA induced in rat brain by LPS challenge was reduced by intraperitoneal administration of methylprednisolone (2×15 mg kg−1) and of rolipram (3×3 mg kg−1). 7 We suggest that peripheral administration of LPS induces a time‐dependent expression of TNF‐α in rat brain, presumably in microglial cells, and that methylprednisolone and rolipram inhibit LPS‐induced expression of TNF‐α in these cells via a decrease of TNF‐α mRNA stability and/or TNF‐α gene transcription.

Recovery and brain reorganization after stroke in adult and aged rats

Stroke is a prevalent and devastating disorder, and no treatment is currently available to restore lost neuronal function after stroke. One unique therapy that improves recovery after stroke is neutralization of the neurite inhibitory protein Nogo‐A. Here, we show, in a clinically relevant model, improved functional recovery and brain reorganization in the aged and adult rat when delayed anti–Nogo‐A therapy is given after ischemic injury. These results support the efficacy of Nogo‐A neutralization as treatment for ischemic stroke, even in the aged animal and after a 1‐week delay, and implicate neuronal plasticity from unlesioned areas of the central nervous system as a mechanism for recovery. Ann Neurol 2005;58:950–953

The Second-Generation Active Aβ Immunotherapy CAD106 Reduces Amyloid Accumulation in APP Transgenic Mice While Minimizing Potential Side Effects

Immunization against amyloid-β (Aβ) can reduce amyloid accumulation in vivo and is considered a potential therapeutic approach for Alzheimers disease. However, it has been associated with meningoencephalitis thought to be mediated by inflammatory T-cells. With the aim of producing an immunogenic vaccine without this side effect, we designed CAD106 comprising Aβ1–6 coupled to the virus-like particle Qβ. Immunization with this vaccine did not activate Aβ-specific T-cells. In APP transgenic mice, CAD106 induced efficacious Aβ antibody titers of different IgG subclasses mainly recognizing the Aβ3–6 epitope. CAD106 reduced brain amyloid accumulation in two APP transgenic mouse lines. Plaque number was a more sensitive readout than plaque area, followed by Aβ42 and Aβ40 levels. Studies with very strong overall amyloid reduction showed an increase in vascular Aβ, which atypically was nonfibrillar. The efficacy of Aβ immunotherapy depended on the Aβ levels and thus differed between animal models, brain regions, and stage of amyloid deposition. Therefore, animal studies may not quantitatively predict the effect in human Alzheimers disease. Our studies provided no evidence for increased microhemorrhages or inflammatory reactions in amyloid-containing brain. In rhesus monkeys, CAD106 induced a similar antibody response as in mice. The antibodies stained amyloid deposits on tissue sections of mouse and human brain but did not label cellular structures containing APP. They reacted with Aβ monomers and oligomers and blocked Aβ toxicity in cell culture. We conclude that CAD106 immunization is suited to interfere with Aβ aggregation and its downstream detrimental effects.