Postmortalna imunohistohemijska dijagnostika difuzne aksonske lezije u slučajevima kraniocerebralnog povređivanja

Abstract

Difuzna aksonska lezija (DAL) je veoma čest ali nedovoljno istražen tip kraniocerebralne traume, sa značajnim sudskome-dicinskim konsekvencama. Osnovni biomehanički princip njenog nastanka je akceleracijsko-deceleracijski mehanizam sa elemen-tima rotacije glave, što uzrokuje istezanje i smicanje pojedinih slojeva moždanog tkiva. Destrukcija aksonskog citoskeleta prekida aksoplazmatski transport i uzrokuje nakupljanje trans-portovanog materijala na mjestima aksonskih oštećenja. Odgovarajućim dijagnostičkim tehnikama (npr. imunohisto-hemijskim bojenjem na beta amiloid prekursor protein – βAPP) vizualizuje se kao nepravilna aksonska izvijuganost i zadebljanja oštećenih/prekinutih aksona. Najčešća distribucija ovih prom-jena je uz središnje cerebralne strukture: parasagitalna bijela masa cerebruma, kapsula interna, korpus kalozum, forniks, rostralni dijelovi moždanog stabla i dr. Ciljevi studije; Utvrditi prisustvo DAL-a u slučajevima nadživljavanja kraćeg od dva sata. Ispitati učestalost i topografsku distribuciju aksonske lezije, njenu povezanost s drugim vrstama tupe kraniocerebralne traume i udruženost sa traumatskim cerebralnim mikrokrvarenjima. Materijal i metode: Kao materijal za ovo prospektivno istraži-vanje poslužilo je moždano tkivo 36 smrtno stradalih osoba u akceleracijsko-deceleracijskim mehanizmima, podijljen u dvije grupe prema dužini nadživljavanja: 17 umrlih < 2 sata i 19 umrlih ≥ 2 sata. Uzimani su uzorci moždanog tkiva iz parasagi-talne bijele mase frontalnog režnja, genu i splenium korpus kalozuma, rostralnog dijela ponsa. Imunohistohemisjkim bojenjem registrovana je βAPP pozitivnost analiziranih isječaka i semikvantitativno procjenjena (skala po Gentleman-u) a za gradi-ranje težine DAL-a korištena je klasifikacija po Adamsu. Za testiranje razlika u raspodjeli atributivnih obilježja korišten je χ2 test a Kruskal-Wallis testom razlike u raspodjeli varijabli sa ordinalnom skalom mjerenja između nezavisnih varijabli. Za analizu povezanosti dva obilježja sa ordinalnom skalom posluži-la je Spirmanova neparametrijska korelacija. Rezultati: Akosnska lezija utvrđena je u 88,9% slučajeva ukupnog uzorka (82,3% u grupi nadživljavanja < 2 sata, 94,7% u grupi nadživljavanja ≥ 2 sata). Najkraće vrijeme nadživljavanja sa potvrđenom βAPP ekspresijom iznosilo je 20-25 minuta, u tri slučaja. Registrovana je prilično ravnomjerna zastupljenost aksonske lezije kroz posmatrane regije mozga uz pomjeranje incidence i jačine βAPP ekspresije ka zadnjim strukturama moždanog tkiva i najveća učestalost jako izražene imunopo-zitivnosti u ponsu. Spirmanov test neparametrijske korelacije potvrdio je značajnu povezanost između βAPP pozitivnosti u parasagitalnoj bijeloj masi frontalnog režnja i genu korpus kalozuma (ρ=0,395) i između splenium korpus kalozuma i ponsa (ρ=0,627). ustanovljena je povezanost ekspresije βAPP imunopo-zitivnosti u genu korpus kalozuma sa prisustvom petehijalnih krvarenja u bijeloj masi frontalnog režnja (p=0,023) te βAPP imunopozitivnosti u ponsu sa interhemisferično-paramezen-cefaličnom subarahnoidalnom hemoragijom (p=0,035). χ2 test potvrdio je povezanost traumatskih mikrokrvarenja u genu korpus kalozuma sa βAPP imunopozitivnošću u sve četiri posmatrane regije mozga, uz najizraženiju vezu upravo u genu korpus kalozuma (p=0,011). Kada se podaci grupišu (prisutna/nije prisutna aksonska lezija), χ2 test potvrdio je značajnu vezu između prisustva aksonske lezije i mikrokrvarenja u bijeloj masi frontalnog režnja (p=0,028), kao i genu korpus kalozuma (p=0,004). Nije potvrđena značajna razlika u težini aksonske lezije (po Adamsu) između grupa kao ni u βAPP ekspresivnosti po analiziranim lokalizaci-jama moždanog tkiva između grupa. Zaključci: Imunohistohemijsko bojenje na βAPP marker pokazalo se visoko efikasnim u dokazivanju traumatske aksonske lezije kod nadživljavanja kraćeg od 2 sata. Nema značajne razlike u distribu-ciji aksonske lezije kroz posmatrane središnje regije mozga, uz primjetnu jaču βAPP ekspresivnost u ponsu. Potvrđena je pozitivna korelacija između prisutva aksonske lezije u ponsu i interhemisferično-paramezencefalično lokalizovane subara-hnoidalne hemoragije. Nađena pozitivna korelacija između prisustva mikrohemoragija i βAPP pozitivnosti u genu korpus kalozuma u skladu je sa aktuelnim stavovima da postoji određena veza između traumatske cerebralne mikrohemoragije i aksonske lezije u mediosagitalnim regijama mozga.

Introduction: Diffuse axonal lesion (DAL) is a very common but understudied type of craniocerebral trauma, with significant forensic consequences. The basic biomechanical principle of its origin is an acceleration-deceleration mechanism with elements of head rotation, which causes stretching and shearing of certain layers of brain tissue. Destruction of the axonal cytoskeleton interrupts axoplasmic transport and the accumulation of transported material at the sites of axonal damage. With appropriate diagnostic techniques (eg immunohistochemical staining for beta-amyloid precursor protein - ßAPP) it is visualized as irregular axonal tortuosity and thickening of damaged/broken axons. The most common distribution of these changes is along the central cerebral structures: parasagittal white mass of the cerebrum, internal capsule, corpus callosum, fornix, rostral parts of the brainstem, etc. Aims of the research: Determine the presence of DAL in cases of survival of less than two hours. To examine the frequency and topographical distribution of axonal lesions, its association with other types of blunt craniocerebral injury, and an association of DAL with traumatic cerebral microbleeds. Material and methods: The material for this prospective research was the brain tissue of 36 persons who died in acceleration-deceleration mechanisms, divided into two groups according to the length of survival: 17 died in a time period of less than 2 hours and 19 died in a time period of more than or equal to 2 hours. Brain tissue samples were taken from the parasagittal white matter of the frontal lobe, the genu and splenium of the corpus callosum, and the rostral part of the pons. Immunohistochemical staining registered ßAPP positivity of the analyzed sections and semiquantitatively assessed according to the Gentleman scale, while grading of the severity of DAL was done according to the Adams classification. The χ2 test was used to test differences in the distribution of attributive characteristics, and the Kruskal-Wallis test was used to test differences in the distribution of variables with an ordinal measurement scale between independent variables. The analysis of the association of two characteristics with an ordinal scale was performed using Spearman's non-parametric correlation. Results: The presence of an axonal lesion was determined in 88.9% of cases of the total sample (in the group of survival less than 2 hours in 82.3%, and in the group of survival more than or equal to 2 hours in 94.7% of cases). The shortest survival time with confirmed βAPP expression was 20 - 25 minutes, in three cases. A fairly even distribution of axonal lesions was registered throughout the observed brain regions with a shift in the incidence and intensity of βAPP immunoexpression towards the posterior structures of the brain tissue and the highest frequency of strong immunopositivity in the pons region. Spearman's non-parametric correlation test confirmed a statistically significant correlation between βAPP immunopositivity in the parasagittal white matter of the frontal lobe and the genu of the corpus callosum (ρ=0.395) and between the splenium of the corpus callosum and the pons (ρ=0.627). There was a correlation between the expression of βAPP immunopositivity in the anterior segment of the corpus callosum (genu) and the presence of petechial hemorrhages in the white matter of the cerebrum (p=0.023), and the expression of βAPP immunopositivity in the pons with interhemispheric-paramesencephalic subarachnoid hemorrhage (p=0.035). The χ2 test confirmed the association of traumatic microhemorrhages in the anterior part of the corpus callosum with βAPP immunopositivity in all four observed brain regions, with the most pronounced connection precisely in the corpus callosum genu (p=0.011). When data are grouped (axonal lesion present/absent), the χ2 test confirmed a significant association between the presence of an axonal lesion and microbleeds in the white matter of the frontal lobes (p=0.028), as well as the genu of the corpus callosum (p=0.004). No statistically significant difference was confirmed between the groups in the severity of the axonal lesion (according to Adams) nor in the βAPP expressivity by the analyzed brain tissue localizations. Conclusions: Immunohistochemical staining for the βAPP marker proved to be highly effective in proving the presence of traumatic axonal lesions in a survival period of less than two hours. There is no significant difference in the distribution of the axonal lesion through the observed central regions of the brain, with a noticeable stronger βAPP expressivity in the pons. A positive correlation was confirmed between the presence of an axonal lesion in the pons and interhemispheric-paramesencephalically localized subarachnoid hemorrhage. The positive correlation found between the presence of microhemorrhages and βAPP immunopositivity in the anterior aspects of the corpus callosum is consistent with the current views that there is most likely a certain connection between traumatic cerebral microhemorrhages and axonal lesions in the mediosagittal regions of the brain.