The total dose administered (8
The total dose administered (8.64 g) was identical to Study A. seen in the ipsilateral cortex, thalamus and hippocampus of brain-injured animals, and anti-MAG mAb was detectable in the hippocampus, fimbria and ventricles. Brain-injured animals receiving anti-MAG mAb showed significantly improved recovery of sensorimotor function at 6 and 8 weeks (< 0.01) post-injury when GSK2110183 analog 1 compared with brain-injured IgG-treated animals. Additionally, at 8 weeks post-injury, the anti-MAG mAb-treated brain-injured animals demonstrated significantly improved cognitive function and reduced hemispheric tissue loss (< 0.05) when compared with their brain-injured controls. These results indicate that MAG may contribute to the pathophysiology of experimental TBI and treatment strategies that target MAG may be suitable for further evaluation. and evidence suggests that inhibitors of axonal growth present in myelin, such as Nogo-A, oligodendrocyte-myelin glyco-protein and myelin-associated glycoprotein (MAG), may prevent axonal outgrowth in models of nervous system injury such as cerebral ischemia, traumatic spinal cord injury and peripheral nerve injury (Caroni have been restricted to adult neurons (McKerracher neutralization of a soluble form of MAG (dMAG) resulted in an increase in GSK2110183 analog 1 neurite outgrowth (Tang immediately post-optic nerve crush injury has been shown to improve regeneration of the optic nerve tract (Wong = 59) was induced as originally described by McIntosh = 43) received anesthesia and all surgical procedures without FP brain injury. The Luer-Lok fitting was then removed and the incision sutured. Animals were placed on heating pads from the initiation of anesthesia until 60 min post-pump implantation in order to maintain normothermia. Pump implantation and intracerebroventricular drug administration At 1 h post-injury, surviving animals were randomized to receive an intracerebroventricular injection of either 0.12 mg/mL inhibitory anti-MAG mAb (72 L; a kind gift from Glaxo Smith Kline, antibody originally from Chemicon, Hampshire, UK, with additional preparation as per Irving = 6) or control IgG mAb (= 5). Sham-injured controls similarly received either anti-MAG mAb (= 8) or control IgG (= 6). At 72 h post-injury, animals were overanesthetized with sodium pentobarbital (75 mg/kg) and transcardially perfused with heparinized saline followed by 4% paraformaldehyde. The brains were removed and post-fixed overnight at 4 C in paraformaldehyde, and were then transferred into 30% sucrose solution for 3?4 days, snap frozen in 2-methylbutane at ?20 C, and stored at ?80 C. Brains were cut on a freezing microtome into 40-m free-floating sections. Detection of anti-myelin-associated glycoprotein monoclonal antibody or control antibody Following blocking for 1 h with 3% normal horse serum, donkey anti-mouse IgG biotin (1 : 1000; Jackson ImmunoResearch, West Grove, PA, USA) was applied to every 12th section from Bregma ?1.3 to ?7.3. The initial Plat order was determined in a random fashion. Following an overnight incubation at 4 C, the avidin-biotin peroxidase method (Vector Laboratories, Burlingame, CA, USA) was used for visualization of the drug or control antibody. Internal controls included use of non-antibody-treated tissue sections and omission of secondary antibody from the protocol. Expression of myelin-associated glycoprotein post-injury Following blocking for 1 h with 3% normal horse serum, goat anti-MAG (1 : 2000; R and D Systems, Abingdon, UK) was applied to every 12th section from Bregma ?1.3 to ?7.3. The initial section chosen was adjacent to that chosen for drug diffusion. Following an overnight incubation at 4 C, sections were washed and incubated in biotinylated donkey anti-goat IgG (Jackson ImmunoResearch) at a concentration of 1 1 : 1000. Following the 1-h secondary antibody incubation period, the avidin-biotin-peroxidase method was used for visualization of MAG within the brain sections. Internal controls included deletion GSK2110183 analog 1 of the primary antibody from the GSK2110183 analog 1 protocol. Study B. Evaluation of neurobehavioral function and tissue loss To examine the long-term neurobehavioral effects of anti-MAG mAb following TBI, brain-injured animals were randomized to receive either the inhibitory anti-MAG mAb (= 25) or control antibody (= 20, = 14) or anti-MAG mAb (= 15). The total dose administered (8.64 g) was identical to Study A. Following surgery or injury, neurological motor function was evaluated for 2 months in surviving animals in sham-injured (control-treated = 13 and anti-MAG mAb-treated = 13) and brain-injured (control-treated = 17 and anti-MAG mAb-treated = 18) rats using a battery of functional tests which have been.