| Objectives:1. To establish an animal model of open spinal cord injury with seawater immersion inrats. Observed the early influence of seawater immersion on the function of nerves andpathological changes after open spinal cord injury with seawater immersion in rats, toevaluate the reliability of the model.2. By observing the long-term changes in motor function and motor evoked potential s(MEP) in rats and the modification of spinal cord blood flow in certain period, to study thelong-term effects of seawater immersion on the function of spinal cord and the changes oflocal spinal cord blood flow. To explore the protection effects on neurological function andspinal cord blood flow by early application Lipo PGE1intravenous therapy.3. Through detecting neuronal apoptosis index and apoptosis-related protein Bcl-2,Bax expression of the specimens of injured spinal cord, to study the effects of seawaterimmersion on nerve cell apoptosis after spinal cord injury and its mechanisms. Exploredthe anti-apoptotic effect of Lipo PGE1by early intravenous administration.Methods:1. In the first part of the experiment, adult healthy maleSprague-Dawley（SD） ratswere randomly divided into six groups: group A(sham), group B (open spinal cord injury),group C (open spinal cord injury with normal saline soak30min), group D (open spinalcord injury with normal saline soak60min), group E (open spinal cord injury withseawater immersion30min) and group F (open spinal cord injury with seawater immersion60min). The rat models of open SCI were build based on modified Allen’ s method. Theneurological function of each group rats weremeasured using BBB locomotor rating scalesand Rivlin’s incline plane test before the experiment and at3h,6h,12h,24h,72h followingthe intervention. Morphological and pathological changes of specimens of injured spinalcord were observed generally and by HE staining microscopyrespectively.2. In the second part of the experiment, SD rats were randomly divided into Shamgroup, SCI group, seawater immersion (SW) group and Lipo PGE1group. The behavior ofeach group rats were assessed with BBB locomotor rating scales and Rivlin’s incline planetest before surgery and at24h,72h,1w,2w,4w,8w after the intervention to compare thedifference between the each groups; The motor evoked potentials (MEP) were detectedbefore injury and at0h,24h,72h,1w,2w,4w,8w, respectively after soaking to observeand record the latency and amplitude changes; The blood flow changes of the dorsal spinal cord were monitored using laser speckle contrast imaging (LASCI) system prior to injuryand at0h after injury,0h,1h,2h,3h,6h,12h,24h,72h after soaking.3. In the third part of the experiment, SD rats were still randomly divided into Shamgroup, SCI group, SW group and Lipo PGE1group. The neuronal apoptosis was observedby TUNEL method, and the expression of apoptosis related protein Bcl-2and Bax weredetected using the SABC immunohistochemical assay at3h,6h,12h,24h,72h and1wrespectively following the intervention.Results:1. After SCI the BBB scores and function angle values of Rivlin’s incline plane test ofeach group rats were significantly decreased. Group E, F were not significantly differentcompared with group B, C, and D at each time point before72h, however, at the72h timepoint, E, F group were significantly lower than B, C, D group, the difference wassignificant statistically, furthermore group F was significantly lower than group E, incontrast, group B, C, and D had no significant difference bypairwise comparison;After SCIeach group showed pathological lesions, such as spinal cord edema, hemorrhage,inflammation and necrosis. These pathological damages were obsereved in pure SCI group(group B) peaked at12h, and the pathological damages of seawater immersion groups(Group E, F) were relatively light at3h and6h time point, but at12h,24h and72h, theabove-mentioned pathological damages rapidly increasing, in addition the degree had beensignificantly heavier than group B and normal saline immersion groups (group C, D).Group F vs. Group E, the pathological damage of Group F was moresevere. There was nosignificant difference was observed in group B, C, and D at each time point.2. After SCI the BBB scores and function angle values of Rivlin’s incline plane test ofeach group rats were significantly decreased, and subsequently recovered slowly, butobserved to8w still significantly lower compared with the preoperative level. At24h eachgroup rats had no significant differences after SCI by pairwise comparison. At each timepoint from72h to8w, SW group was significantly lower than SCI group; Lipo PGE1groupwas significantly higher than SW group and SCI group. After SCI MEP signalsdisappeared, at24h time point, MEP signals can be recorded again, mainly showed aslatency prolonged and the amplitude decreased, and subsequently restored in each group.Each group was observed to8w their latency and amplitude were unable to return topre-injury level, at24h each group rats showed no significant differences in latency andamplitude after SCI bypairwise comparison. At each time point from72h to8w, SW group compared with SCI group, the latency was significantly prolonged and the amplitudesignificantly decreased, compared with SW group and SCI group, the latency wassignificantly shortened and the amplitude was significantly increased in Lipo PGE1.Immediately after SCI the velocity of spinal cord blood flow (SCBF) was significantlydecreased in each group rats, and no significant difference between the groups, and then,SW group and Lipo PGE1group rats according to the experimental requirements weregiven seawater immersion and administration, the SCBF velocity of these two groupsdeclined further immediately after immersion, between the two groups had no significantdifference. Meanwhile, the SCBF velocity of SCI group was slightly restored, and hadbeen significantly higher than the above two groups. At1h and2h time point, the SCBFvelocity in each group were recovered, the three groups were significantly different bypairwise comparison at1h (SCI group> Lipo PGE1group> SW group),to2h Lipo PGE1group just less below SCI group and had no significant difference with it, meantime, theimprovement of SW group was slowly, had been significantly lower than the above twogroups. However the SCBF velocity of each group dropped again at3h, to6h SCI groupand SW group continued to decline slightly, while Lipo PGE1group improvedsignificantly, and had been significantly higher than SCI group, the three groups showedsignificant differences (Lipo PGE1group> SCI group> SW group). The SCBF velocity ineach group was recovered continually, observed to72h each group was unable to restore tothe level before the injury.3. Apoptotic cells were significantly increased in the rat spinal cord at3h after SCI,and showed an upward trend, the apoptotic index (AI) were highest at24h, and then beganto decline, a lot of apoptotic cells still could be detected until1w (SW group> SCI group>Lipo PGE1group). At3h and6h time point, the AI showed no significant differencebetween SW group and Lipo PGE1group, and were significantly lower than SCI group,and at each subsequent time point the AI of SW group was significantly higher than LipoPGE1group and SCI group, in addition Lipo PGE1group was significantly lower than SCIgroup. The expressions of Bcl-2and Bax were significantly increased in three injuredgroups after SCI, and showed an upward trend. Bcl-2expression in each group reached thepeak at24h, while the expression peak of Bax was at12h, and then both began to decline,to1w the expressions of them were still high. The expression of Bcl-2in SW group wassignificantly lower than SCI group at each time point, whereas the expression of Bax wasrather special, at3h and6h time points showed significantly lower than SCI group, but significantly higher than it at each subsequent time point. The expression of Bcl-2in LipoPGE1group was significantly higher than SW group and SCI group at all time, but theexpression of the Bax was lower than the two groups.Conclusions:1. The animal model of open spinal cord injury with seawater immersion in rats isstable and reliable. Seawater immersion could aggravate the neurological function of ratsafter open SCI, and the longer soaking time the greater the impact; Seawater immersionearly could delay and reduce pathological lesions of spinal cord injury, such as edema,hemorrhage, inflammation and necrosis, but ultimately the level of the above-mentionedpathological damages were heavier than pure SCI.2. Seawater immersion could cause long-term impact on neurological function afteropen spinal cord injury, was detrimental to their recovery, these results has been confirmedby behavioral observations and electrophysiological tests; After SCI the velocity of spinalcord blood flow would be significantly reduced, seawater immersion could aggravate thisdamage and prolong their recovery time; Early application of Lipo PGE1intravenoustherapy could improve nerve function and spinal cord blood flow velocity after open spinalcord injury with seawater immersion, and promote their recovery.3. Seawater immersion could increase the expression of neuronal apoptosis relatedprotein Bcl-2and Bax, but compared with single injury in rats, the expression of Bcl-2wasless and Bax expression was significantly higher, thereby promoted neuronal apoptosis;Early application of Lipo PGE1intravenous therapy could increase the expression of Bcl-2,decrease the expression of Bax, accordingly inhibit neuronal apoptosis. |
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