| Objective: 1. To observe ultrastructural changes of axons after the stretch injury and discuss pathological significances of ultrastructural abnormalities of injured axons.2. To investigate the expressions of HSP70 in neurons and oligodendrocytes after the stretch injury and the effects of heat stress on HSP70 expression and explore neuroprotective effect of HSP70 on axonal injury.Methods: The experimental studies included two parts.The first part of the studies is ultrastructural study on axonal injury in DAI model 15 male Wistar rats were randomly divided into control group with 3 animals and experimental group with 12 animals. Animals in experimental group were subjected to stretch injury to the right optic nerves. Three animals from experimental group were killed at 2h, 4h, 24h and 7d after injury respectively. The ultrastructural changes of injured optic nerves were examined under a transmission electronmicroscope.The second part of the studies is the experimental study on protective effect of heat stress on axonal injury57 male Wistar rats were randomly divided into four groups: control group (Group A with 3 animals), stretch-only group(Group B with 18 animals), heat stress-only group(Group C with 18 animals) and heat stress pretreatment plus stretch group(Group D with 18 animals). Stretch injury was induced in the right optic nerves of rats in Groups B. Heat stress was applied to animals of Group C. Animals in Group D was subjected to the same stretch injury as Group B after sustaining the same heat stress as Group C. Three animals from each group (Group B, Group C and Group D) were killed at 4h, 8h, 16h, 1d, 3d, 5d respectively. Morphological changes of optic nerves, retinal ganglion cells (RGCs) and oligodendrocytes (OLs) in optic nerves after stretch injury were examined under light microscope and expressions of HSP70 in RGCs and OLs after heat stress or/and stretch injury were observed using immunohistochemistry. Expressions of HSP70 were represented by optical density (OD). Student t-test was used for statistic analysis.Results: 1. Ultrastructural study on axonal injury in DAI model:Prominent ultrastructural changes of axons in optic nerves occurred after stretch injury. The subcellular abnormalities of injured axons included dislocations of myelin sheath, protrusions of nodal axolemma, reduction in number of microtubules or even loss of microtubules, compaction of neurofilaments and swelling of mitochondria. Two distinct pathological changes were identified and accordingly the injured axons were classified into two groups, one of which demonstrated axonal swelling and the other axonal constriction. The axons with axonal swelling occurred at acute stage (2h～4h) of injury and were characterized by a focal increase in axonal diameter, accumulation of membranous organelles, dissolution of axonal cytoskeleton and thinning of myelin sheath. The axons with axonal constriction were found from 2h to 7d after injury and characterized by a focal reduction in axonal diameter, occurence of enlarged periaxonal space, compaction of neurofilaments, maintenance of stable neurofilament network, shrink of myelin sheath.2. The experimental study on protective effect of heat stress on axonal injury:(1) Pathological Observation: Pathological changes of axons, RGCs, OLs after stretch injury to the optic nerves were identified morphologically or quantitatively, and significantly ameliorated by pre-treatment with heat stress before stretch injury. (2) Expression of HSP70 in RGCs and OLs: ①RGCs. Group A showed weak expression of HSP70 in both cytoplasm and nucleus of RGCs (OD: 0.18±0.04). An increased expression of HSP70 in Group B occurred at every time point with a peak at 1d (0.28±0.03) . Expression of HSP70 in Group C also increased significantly except at 5d. A much more enhanced expression HSP70 were found in Group D with an earlier peak ( at 16h,0.31±0.05) compared with Group B and C and longer period compared with Group...
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