Metabonomics Analysis of Brain Stem Tissue in Rats with Primary Brain Stem Injury Caused Death / 法医学杂志

OBJECTIVES@#To explore the potential biomarkers for the diagnosis of primary brain stem injury (PBSI) by using metabonomics method to observe the changes of metabolites in rats with PBSI caused death.@*METHODS@#PBSI, non-brain stem brain injury and decapitation rat models were established, and metabolic maps of brain stem were obtained by LC-MS metabonomics method and annotated to the HMDB database. Partial least square-discriminant analysis (PLS-DA) and random forest methods were used to screen potential biomarkers associated with PBSI diagnosis.@*RESULTS@#Eighty-six potential metabolic markers associated with PBSI were screened by PLS-DA. They were modeled and predicted by random forest algorithm with an accuracy rate of 83.3%. The 818 metabolic markers annotated to HMDB database were used for random forest modeling and prediction, and the accuracy rate was 88.9%. According to the importance in the identification of cause of death, the most important metabolic markers that were significantly up-regulated in PBSI group were HMDB0038126 (genipinic acid, GA), HMDB0013272 (N-lauroylglycine), HMDB0005199 [(R)-salsolinol] and HMDB0013645 (N,N-dimethylsphingosine).@*CONCLUSIONS@#GA, N-lauroylglycine, (R)-salsolinol and N,N-dimethylsphingosine are expected to be important metabolite indicators in the diagnosis of PBSI caused death, thus providing clues for forensic medicine practice.

Propentofylline reduces glial scar development following gliotoxic damage in the rat brainstem / Propentofilina reduz o desenvolvimento da cicatriz glial após dano gliotóxico no tronco encefálico de ratos

ABSTRACT Propentofylline is a xanthine derivative that depresses activation of glial cells, whose responses contribute to neural tissue damage during inflammation. Ethidium bromide injection into the central nervous system induces local oligodendroglial and astrocytic loss, resulting in primary demyelination, neuroinflammation and blood-brain barrier disruption. Surviving astrocytes present a vigorous reaction around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). Objective This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. Method Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. Results and Conclusion Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury.

RESUMO A propentofilina é uma xantina que deprime a ativação das células gliais, cujas respostas contribuem para o dano neural durante inflamação. A injeção de brometo de etídio no sistema nervoso central induz a perda oligodendroglial e astrocitária, resultando em desmielinização, neuroinflamação e ruptura da barreira hematoencefálica. Os astrócitos sobreviventes apresentam vigorosa reação ao redor da lesão com aumento da imunorreatividade à proteína glial fibrilar ácida (GFAP). Objetivo Este estudo objetivou avaliar o efeito da propentofilina sobre a resposta astrocitária após injúria gliotóxica. Método Ratos Wistar foram injetados com brometo de etídio na cisterna basal e tratados ou não com propentofilina (12.5mg/kg/dia, intraperitoneal). Amostras do tronco encefálico foram coletadas dos 15 aos 31 dias pós-injeção do gliotóxico e processadas para estudo ultraestrutural e imuno-histoquímico para GFAP. Resultados e Conclusão Os resultados demonstram que a propentofilina reduziu a ativação astrocitária até o 21o dia, sugerindo que essa droga pode atuar na redução da cicatriz glial após injúria.

Proteomic Analysis of Rat Brain Stem with DAI by MALDI-TOF-MS / 法医学杂志

OBJECTIVE@#To establish a diagnostic model for diffuse axonal injury (DAI) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To screen the proteins or peptides associated with DAI for providing the biomarkers with theoretic foundation.@*METHODS@#Fifteen male Sprague-Dawley rats were randomly divided into DAI group (n = 10) and control group (n = 5). The protein or peptide expression profiles of rat brain stem were detected by MALDI-TOF-MS. ClinProTools 2.2 software was used to find specific peaks, and a diagnostic model was established by the genetic algorithm.@*RESULTS@#There were significant differences in 61 peaks of DAI group (P < 0.05), 9 peaks were down-regulated and 52 up-regulated. The diagnostic model was established based on 5 different peaks. The specificity and sensitivity of cross validation was 96.14% and 95.98%; while the specificity and sensitivity of blind validation showed was 73.33% and 70.00%, respectively.@*CONCLUSION@#A specific and sensitive diagnostic model of DAI can be established by MALDI-TOF-MS to provide a potential value for determining DAI in forensic practice.

Expression of S100B and GFAP after primary brainstem injury in rat / 法医学杂志

OBJECTIVE@#To study the expression of S100B and glial tibrillory acidic protein (GFAP) atter primary brainstem injury in rat and discuss the changes with brainstern injury time and their mechanism in the injury.@*METHODS@#The brainstem injury animal model was established using the mechanical impacting method. The HE staining, Gless argentaffin staining and SP immunohistochemical method were applied to observe the changes of S100B and GFAP at different injury time. The immunostaining results were measured statistically with imaging analysis technology.@*RESULTS@#A large number of S100B positive cells could be seen in 30 min. Afterward, expression increased gradually with time and peaked up in 24 h, and reversed back the normal in 72h. The GFAP positive cells showed rise continually in 30 min, and reached the peak in 48 h, then started to decrease, but still higher than that in control.@*CONCLUSION@#The expression of S100B and GFAP is correlated with post traumatic intervals after brainstem injury in rat, and may be useful in estimation post traumatic intervals and nerve regeneration.

Expression of c-jun protein after experimental rat brain concussion / 法医学杂志

OBJECTIVE@#To observe e-jun protein expression after rat brain concussion and explore the forensic pathologic markers following brain concussion.@*METHODS@#Fifty-five rats were randomly divided into brain concussion group and control group. The expression of c-jun protein was observed by immunohistochemistry.@*RESULTS@#There were weak positive expression of c-jun protein in control group. In brain concussion group, however, some neutrons showed positive expression of c-jun protein at 15 min after brain concussion, and reach to the peak at 3 h after brain concussion.@*CONCLUSION@#The research results suggest that detection of c-jun protein could be a marker to determine brain concussion and estimate injury time after brain concussion.

Tissue-specific activation of mitogen-activated protein kinases for expression of transthyretin by phenylalanine and its metabolite, phenylpyruvic acid

Phenylketonuria is an autosomal recessive disorder caused by a deficiency of phenylalanine hydroxylase. Transthyretin has been implicated as an indicator of nutritional status in phenylketonuria patients. In this study, we report that phenylalanine and its metabolite, phenylpyruvic acid, affect MAPK, changing transthyretin expression in a cell- and tissue-specific manner. Treatment of HepG2 cells with phenylalanine or phenylpyruvic acid decreased transcription of the TTR gene and decreased the transcriptional activity of the TTR promoter site, which was partly mediated through HNF4alpha. Decreased levels of p38 MAPK were detected in the liver of phenylketonuria-affected mice compared with wild-type mice. In contrast, treatment with phenylalanine increased transthyretin expression and induced ERK1/2 activation in PC-12 cells; ERK1/2 activation was also elevated in the brainstem of phenylketonuria-affected mice. These findings may explain between-tissue differences in gene expression, including Ttr gene expression, in the phenylketonuria mouse model.

Induction of Fos protein immunoreactivity by spinal cord contusion

The objective of the present study was to identify neurons in the central nervous system that respond to spinal contusion injury in the rat by monitoring the expression of the nuclear protein encoded by the c-fos gene, an activity-dependent gene, in spinal cord and brainstem regions. Rats were anesthetized with urethane and the injury was produced by dropping a 5-g weight from 20.0 cm onto the exposed dura at the T10-L1 vertebral level (contusion group). The spinal cord was exposed but not lesioned in anesthetized control animals (laminectomy group); intact animals were also subjected to anesthesia (intact control). Behavioral alterations were analyzed by Tarlov/Bohlman scores, 2 h after the procedures and the animals were then perfused for immunocytochemistry. The patterns of Fos-like immunoreactivity (FLI) which were site-specific, reproducible and correlated with spinal laminae that respond predominantly to noxious stimulation or injury: laminae I-II (outer substantia gelatinosa) and X and the nucleus of the intermediolateral cell column. At the brain stem level FLI was detected in the reticular formation, area postrema and solitary tract nucleus of lesioned animals. No Fos staining was detected by immunocytochemistry in the intact control group. However, detection of FLI in the group submitted to anesthesia and surgical procedures, although less intense than in the lesion group, indicated that microtraumas may occur which are not detected by the Tarlov/Bohlman scores. There is both a local and remote effect of a distal contusion on the spinal cord of rats, implicating sensory neurons and centers related to autonomic control in the reaction to this kind of injury.

Effects of L-arginine on picrotoxin-induced increase in brain ammonia concentrations and convulsions in rats.

The effect of L-arginine (840 mg/kg) pre- (30 min before challenge) and post-treatment (5 min after challenge) period was tested on picrotoxin-induced increase in ammonia concentrations in brain regions (cerebral cortex, brain stem and cerebellum) and the accompanying convulsive responses in adult male rats. The combined effect of L-arginine and diazepam was also tested against picrotoxin-induced convulsions. Picrotoxin-induced increase in ammonia was reverted partially by L-arginine pretreatment. However, L-arginine pretreatment did not show anticonvulsant effect independently or concurrently with diazepam. On the other hand, L-arginine post-treatment reverted ammonia to control level in all brain regions. A partial but significant inhibition of convulsion responses was found in these animals. The combined effect of diazepam and L-arginine post-treatment was much greater than that produced by these agents independently. These findings suggest that ammonia has a partial but significant participation in the convulsant action of picrotoxin. L-arginine has a potential to revert brain ammonia to control level in picrotoxin-treated animals and thereby it has produced a partial protection. The data further indicate that the duration of action of L-arginine is considerably short and has an additive anticonvulsant action with diazepam.

Immobilisation stress induced analgesia in diabetic rats.

Stress is known to produce analgesia. The pain threshold is altered in diabetes. We studied the effect of 1 hr of immobilisation stress on pain threshold in male Wistar rats. The same effect was tested in streptozotocin induced diabetic rats. The pain threshold of tail flick, vocalisation and vocalisation after discharge increased in the control group after the stress procedure. Significant analgesia was also obtained in diabetic rats, for flick and after discharge pain threshold. However the vocalisation threshold was not altered, probably due to the antagonistic action of glucose on opiate receptor at the level of brain stem.

Major bovine brain stem glycoproteins sugar-specifically recognized by endogenous 14 kDa galactose-binding lectin.

In order to identify the complementary glycoproteins (receptors) that are recognized in bovine brain stem by endogenous 14 kDa galactose-binding lectin (BBL), probable glycoproteins were first selected by affinity chromatography of soluble tissue glycoproteins on Rinicus communis agglutinin (RCA)-Sepharose since this lectin had similar sugar specificity to the endogenous lectin. From Western blot of RCA-binding glycoproteins, the lectin, as its peroxidase conjugate sugar-specifically recognized chiefly an 84 kDa glycoprotein subunit and a few minor subunits. On alkaline pH PAGE of the RCA-binding brain stem glycoproteins, a prominent fast moving protein was separated which, on electroelution and dot blotting, was also recognized by BBL sugar-specifically. This glycoprotein was composed of 55 kDa and 58 kDa subunits as seen by SDS-PAGE and was also immunologically distinct from the 84 kDa subunit. Qualitative test on dot blots of the electroeluted glycoproteins using peroxidase conjugates of plant lectins of varying specificities as well as the human serum anti-alpha-galactoside antibody indicated differences in carbohydrate composition between the 84 kDa subunit and the alkaline PAGE fast moving glycoprotein. Membrane-bound brain stem glycoproteins were not recognized by BBL.

Increased brainstem angiotensin-I levels in chronic 2 KIC hypertensive rats.

A study was undertaken on the role of brainstem renin-angiotensin system in maintenance of hypertension in chronic renovascular hypertensive rats. Hypertension was induced by unilateral renal artery clamping, while the contralateral kidney was left intact (2 KIC). Blood pressure (BP), plasma renin activity (PRA) and brainstem angiotensin (ang-I) levels were measured after 24 days, in hypertensive and sham-operated animals. In separate subgroups of these animals, the effect of intracerebroventricular administration of captopril on the parameters listed was studied. The results showed high ang-I levels in 2 KIC rats as compared to controls (P less than 0.05). Captopril administration (500 micrograms/50 microliters icv) caused a fall in BP and increase in brainstem ang-I levels (P less than 0.01). In control animals, however, captopril produced a rise in BP without any significant change in brainstem ang-I levels. Peripheral plasma renin activity was normal, despite significant sodium retention in 2 KIC rats. The results are suggestive of activation of brainstem renin-angiotensin system (RAS) in chronic 2 KIC hypertension.

Involvement of Central adrenergic mechanisms in the induction of cardiac arrhythmias by aconitine nitrate administered intraventricularly.

Aconitine, 10 mug, administered intraventricularly in cats produced cardiae arrhythmias. Intraventricular administration of phenoxybenzamine, propranolol and practolol abolishes the centrally induced cardiac arrhythmias. Intraventricular reserpinization also abolished these cardiac arrhythmias whereas intraventricular administration of 6-hydroxydopamine and tetrabenazine had no effect. Brain stem noradrenaline probably plays a role in these centrally induced cardiac arrhythmias by aconitine.