Model Establishment And Secondary Injury Study In Dogs With Penetrating Craniocerebral Gunshot Wounds

Paper one Model establishment of penetrating craniocerebral gunshot wounds in dogsObjectivesTo establish a new model of penetrating craniocerebral gunshot wounds in dogs, increase the survival time for further research.MethodsNineteen local male adult dogs were randomly divided into 3 groups.Group A for classic model(n=5),shot directly without operation;group B for new model(n=9), which were operated by removing the frontal-temporal-parietal skull before shot at the frontal lobe directly in coronal gyrus;group C for control group(n=5),were operated only.The initial velocity and pellet quality of Dongfeng-SS03 pistol which was used in this experiment was 190～210m/s and 2.60±0.05g accordingly.Thiopentone(25mg/kg) was intraperitoneal injected in dogs as anesthesia before tracheal intubation.The dog head was fixed with left side upward,sandbag and 10-cm-thick board underlying. The pistol was fixed at a distance of 20cm to the dog head,and adjusted by laser indicator.Target point was 2.5～3.0cm backward to the zygomatic process in group A and central section of coronal gyrus in group B.Angle of incidence was 90 degree to the sagittal surface.Local brain tissue was protected with gelatin sponge after being shot.Heart rate(HR),mean artery pressure(MAP) and breath status was recorded by physiological detector.PaO2,PaCO2,HCO3-,serum glucose content,ALT,AST,BUN and Cr were investigated 30 minute after shooting.Six hours later,four parts of brain tissues were saved,which was less then 0.5 cm,0.5 to 2.0 cm,2.0 to 4.0 cm and more than 4.0 cm from the target point in group B and C(in corresponding section).All samples were kept in formalin for routine pathological study and in glutaraldehyde for transmission electronic microscope(TEM) study.ResultThe time of survival and breath pause in group B were longer than group A (p＜0.01).Fluctuating extent of MAP and HR in group B were also bigger than group A. Vacuolization of brain tissue,pericaryon deformation,karyopycnosis,kytoplasm anachromasis,nucleus excursion,horizontal cell obviously enlarged,gitter cell increased,scattered hemorrhage in brain tissue,subarachnoid hemorrhage(SAH), perivascular space obviously enlarged and angiorrhexis could be observed by microscope detection.In TEM study,neuron showed chromatin agglutination in nucleus,nucleolemma ambiguity,kytoplasm dissolve,cell membrane breakage,rough endoplasmic reticulum(RER) distention and degranulation,mitochondrial cristae damage,disappear or vacuolization,ribosome(RI) decrease,lysosome increase.Foot process of gliocyte swelling,medullated nerve fibers demyelinate or collapse and blood brain barrier(BBB) destruction were also detected.The brain tissue near the original wound damaged much severely than that far from it.ConclusionContrasted to the classic model,the new model had a longer survival time, obvious pathological,physiological and ultramicrostructure change,and better concordance of wound. Paper two Mechanism of secondary injury in penetrating craniocerebral gunshot wounds in dogsObjectiveTo investigate the role of TNF-α,COX-2,Caspase-3 and NF-κB/ Rels in secondary injury in penetrating craniocerebral gunshot wounds.MethodsFourteen local male adult dogs were randomly divided into 2 groups.Group B for modified model(n=9),which were operated by removing the frontal-temporal-parietal skull before shot at the frontal lobe directly in coronal gyrus;group C for control group(n=5),were operated only.The initial velocity and pellet quality of Dongfeng-SS03 pistol which was used in this experiment was 190～210m/s and 2.60±0:05g accordingly.Thiopentone(25mg/kg) was intraperitoneal injected in dogs as anesthesia before tracheal intubation.The dog head was fixed with left side upward, sandbag and 10-cm-thick board underlying.The pistol was fixed at a distance of 20cm to the dog head,and adjusted by laser indicator.Target point was 2.5～3.0cm backward to the zygomatic process in group A and central section of coronal gyrus in group B.Angle of incidence was 90 degree to the sagittal surface.Local brain tissue was protected with gelatin sponge after being shot.Six hours after shooting,four parts of brain tissues were saved,which was 0.5 to 2.0 cm,2.0 to 4.0 cm and more than 4.0 cm from the target point in group B and C(in corresponding section),those of group B were marked as group B1,B2 and B3.All samples were kept in formalin for immunohistochemistry(IHC) study and in liquid nitrogen for western blotting and polymerase chain reaction(PCR) study.Gene gapdh was used as internal reference in PCR,and the primers were designed as follow:TNF-α(413bp) upstream primer: 5'CCC CAA GTG ACA AGC CAG TA 3',downstream primer:5'CAA AGT CCA GAT TAG GCA GAT 3';NF-κB(P65)(490bp) upstream primer:5'TAA TCG CCA TAG CCA TAG TCG 3',downstream primer:5'GTT TTG CCT CCC AGT TCT GA 3'; NF-κB(P52)(381bp) upstream primer:5'CCT ATC CAC GAC AAC CTT GC 3', downstream primer:5'CAT AGA TGC TGC TGA CCC AAC 3';COX-2(238bp) upstream primer:5'ATC CCC TTC CTG CGA AAT AC 3',downstream primer: 5'GCA GAA GAA ACT TTT CCA CAA TC 3',Caspase-3(416bp) upstream primer: 5'ACC CGA AGG CTT GCA TAA GG 3',downstream primer:5'ACC GAG GTG CCA TTC CAG TA 3';gapdh(419bp) upstream primer:5'TCC CGC CAA CAT CAA A 3',downstream primer:5' TGA CCT TGC CCA CAG C 3'ResultIHC:TNF-αand COX-2 protein showed positive staining in cytoplasm,and the level of expression ascended gradually from B3 to B1.Both positive rates in group B were higher than that in group C(p＜0.01).Caspase-3 positive staining was detected in cytoplasm and nucleus,group B2 and B3 showed deference from group C(p＜0.01). Western blotting:The relative amounts of TNF-α,COX-2 and Caspase-3 protein in group B were higher than that in group C(p＜0.05).Deference of COX-2 between group B1 and B2 could also be observed.PCR:The relative amounts of TNF-α,P65, P52,COX-2 and Caspase-3 mRNA in group B were higher than that in group C (p＜0.01).And it also showed deference among B1,B2 and B3.Moreover,P65 and COX-2 level ascended gradually from B3 to B1.ConclusionTNF-α,P65,P52,COX-2 and Caspase-3 were all involved in the mechanism of secondary injury in penetrating craniocerebral gunshot wounds.The role of P65 in secondary injury of brain is more important than P52.Neuron degeneration induced by COX-2 may be dominating compared with neuron apoptosis induced by Caspase-3. Paper three Electrocorticography monitoring in penetrating craniocerebral gunshot wounds in dogsObjectiveTo investigate the correlation between penetrating craniocerebral gunshot wounds and ECoG performance by ECoG monitoring in dogs.MethodsLocal male adult dogs were studied as modified model(n=9),which were operated by removing the frontal-temporal-parietal skull before ECoG monitoring,then shot at the frontal lobe directly in coronal gyrus and inspected by ECoG again.The model was established according to the modified method mentioned above.Oxford Medelec system was used in ECoG monitoring.The aciculiform electrodes were set in frontal pole,central region,parietal region and occipital region,with two reference electrodes in scalp.The signal was recorded in reference lead,the sensitivity was 50 uV/5mm,high-frequency filter was 30 Hz,time constant was 0.3 s,paper speed was 30 mm/s,monitored for more than 30 minutes.ResultIn early stage the basic rhythm was generally suppressed and the amplitude was in low level,especially in initial wound region.Both indexes were better in surrounding areas.Amplitude and frequency increased after discontinuously oxygen breathe in.ConclusionECoG monitoring is advantageous in early diagnosis in craniocerebral gunshot wounds.It could be used in the evaluation of injury and the prediction of prognosis.