:Acta NTeurochirurgica
Acta Neurochir (Wien) (1992) 119:153-158
9 Springer-Verlag 1992 Printed m Austria
Pathological Study of Diffuse Axonal Injury Patients Who Died Shortly After Impact T. Yamaki ~, N. Murakami t, Y. Iwamoto 1, Y. Nakagawa 1, S. Ueda 1, Y. Irizawa 2, S. Komura 2, and T. Matsuura 3 Departments of 1Neurosurgery and 2Forensic Medicine, Kyoto Prefectural University of Medicine, and 3Department of Anatomy, Meiji College of Oriental Medicine, Kyoto, Japan
Summary
in 7 cases a n d in o n e o t h e r case 3 d a y s later. F o r
It is generally considered that axonal injury is apparent only on electron microscopy in the very early stage after a closed head injury. To clarify the pathological findings in head injury patients dying very shortly after the impact, we analyzed 8 fatal cases of diffuse axonal injury (DAI) who underwent medicolegal autopsy at the Department of Forensic Medicine of Kyoto Prefectural University of Medicine. Seven cases died within one hour after injury and another one case died 3 days after injury. We studied these cases macroscopically, microscopically, and electron microscopically. Macroscopically all cases showed the typical findings of diffuse axonal injury. Microscopical study of the cases who died within one hour revealed no characteristic findings of DAI such as appearance of retraction bails or microglia. On the other hand, in the case who died only 3 days after injury it showed the typical retraction balls. Electron microscopic study showed the remarkable destruction of cytoskeletal structure of axons in all cases. From our results, it is reasonable to speculate that DAI may be common among head injury patients who die very soon after the impact.
pathological examination, we studied the brains mac-
Keywords:Head injury; early death; diffuse axonal injury; pathological study.
Introduction Clinicopathological reports on human closed head i n j u r y are c o m m o n , b u t p a t h o l o g i c a l f i n d i n g s o f diffuse a x o n a l i n j u r y cases, w h e r e d e a t h o c c u r r e d i m m e d i a t e l y a f t e r t h e i m p a c t , h a v e r a r e l y b e e n r e p o r t e d . It is c o n s i d e r e d t h a t i m m e d i a t e l y f o l l o w i n g t h e initial insult, a x o n a l i n j u r y is o n l y a p p a r e n t b y e l e c t r o n m i c r o s c o p y . H o w e v e r , we c o u l d n o t f i n d a n y p a p e r s p u b l i s h e d o n e l e c t r o n m i c r o s c o p i c f i n d i n g s in the e a r l y stages o f h u m a n a x o n a l injury, a n d o n l y d a t a f r o m e x p e r i m e n t a l m o d e l s s e e m to be a v a i l a b l e 8' 9. 11, 17 T h e r e f o r e , w e s t u d i e d 8 f a t a l cases o f diffuse a x o n a l i n j u r y ( D A I ) . Among them, death occurred within one hour of injury
roscopically, microscopically, and electron microscopically.
Materials and Methods We studied 16 fatal cases of closed head injury and conducted medicolegal autopsies at the Department of Forensic Medicine of Kyoto Prefectural University of Medicine from 1988 to 1989. All of these subjects underwent a complete autopsy and it was confirmed that there were no lesions but those of the brain as the cause of death. Among them in 8 cases, the characteristic findings of DAI 15 were noted macroscopically. Those findings were diffuse subarachnoid haemorrhage, small haemorrhages in subcortical white matter, corpus callosum, basal ganglia, and brain stem. No findings of increased intracranial pressure such as brain swelling and/or large intracerebral haematoma were seen. The interval from death to autopsy of these cases ranged from 4 to 36 hours (Table 1). When the interval from death to autopsy was over 12 hours, the bodies were preserved in a refrigerator. After the autopsy, the whole brain was fixed in 10% formalin. Macroscopic forensic examination was done within one month after the fixation, while microscopic and electron microscopic examinations were done 1-2 years later. For the microscopic examination, representative tissue blocks were obtained from the frontal and temporal cortices, their subcortical white matter, the basal ganglia, the corpus callosum, and the pons. All the blocks were embedded in paraffin and 6 gm sections were cut and stained with haematoxylin-eosin (HE), Luxol fast blue, Bodian's silver impregnation, and toluidine blue. Electron microscopic examination of the corpus callosum and the dorsolateral quadrants of the rostral pons was also done. Specimens fixed in 10% formalin were cut into small pieces and fixed again with 2.5% glutaraldehyde and 1% osmium tetraoxide in 0.1 M phosphate buffer (pH 7.4). After fixation, the specimens dehydrated with graded series of acetone and embedded in Libeak 812. Ultrathin sections were cut on a Reichert Ultracut, stained with uranyl
T. Yamaki etal.: Pathological Study of Diffuse Axonal Injury Patients W h o Died Shortly After Impact
154
Table 1. Clinical Profile of the Autopsy Cases No. Age
Sex
Cause of head injury
Impact site
Skull facture
Autopsy findings (Brain surface)
Intervals from impacts to death
Intervals from death to autopsy
1 2 3 4 5 6 7 8
M M M M M F F M
traffic accident unknown traffic accident traffic accident traffic accident traffic accident traffic accident fall
Rt. Rt. Lt. Rt. Rt. Lt. Rt. Lt.
+ + + + + + + -
SAH, CC SAH SAH, A S H SAH, CC SAH SAH SAH SAH
1h 1h 1h 1h 1h 1h 1h 3d
4h 36 h 10h 32h 11 h 15 h 19 h 24 h
5 18 41 47 50 65 78 19
temporal temporal occipital frontal temporal temporal frontal occipital
S A H subarachnoid haemorrhage, C C cerebral contusion, A S H acute subdural haematoma.
Table 2. Sites of Haemorrhagic Lesions. Findings from the examination of brain slices Case no.
Cortex
Subcortical white matter
I
+
+
+
+
+
2 3 4 5 6 7 8
+ + -. --3/8
+ + + +
-+ + +
+ + + +
-4/8
+ + 7/8
+ + + + + -+ 7/8
.
.
Basal ganglia
Corpus callosum
.
-5/8
Brain stem
acetate and lead nitrate, and examined using a J E D L S-100 electron microscope. Artifacts often affect electron microscopy findings, so, as a control we also studied the corpus callosum and dorsal pons of a 67-year-old w o m a n who died suddenly due to myocardial infarction, a n d underwent a medicolegal autopsy in 1988, which was prepared in the same m a n n e r as the head injury cases.
Results
Clinical Profile of the Autopsy Cases The ages of the cases ranged from 5 to 78 years. Six of the subjects were males and 2 were females. Six cases were injured in traffic accidents, 1 by a fall and 1 by some unknown cause. Four cases were transferred to hospital by an ambulance but 3 of them were "dead on arrival" except for Case 8. Another 4 cases were directly transferred to the Department of Forensic Medicine (Table 1).
Autopsy Findings of the Head Four out of 8 cases had an impact to the temporal region, 2 to the frontal region, and one to the occipital
region. Skull fractures were noted in 7 cases except for Case 8. Intracranial haematoma (a thin acute subdural haematoma) was seen in only one case out of 8 cases. The brain surface showed traumatic subarachnoid haemorrhage in all cases. The sites of intracerebral haemorrhage on cut sections of the brain are shown in Table 2. Cortical haemorrhage was found in 3/8 cases (38%), 5 cases (63%) had it in the subcortical white matter, 4 cases (50%) had basal ganglia haemorrhage, 7 cases (88%) hat it in the corpus callosum, and 7 cases (88%) had brain stem haemorrhage. All of the haemorrhages were small or petechial (Fig. 1). Cerebral contusion was noted in 2 cases (25%). These contusions were small and located on the parasagittal portion of the frontal and parietal lobes.
Microscopic Findings On microscopy, only the haemorrhagic lesions were seen with H E staining. In the cases who died within one hour (cases 1-7) no remarkable axonal changes such as axonal retraction balls (ARBs) were noted in the subcortical white matter, corpus callosum, or dorsolateral quadrants of the rostral pons, in sections stained with both Bodian's silver impregnation and toluidine blue. However, slight reactive axonal swelling was seen in 4 out of 7 cases (57%) (Fig. 2). In Case 8 who survived for 3 days, typical ARBs were noted (Fig. 3).
Electron Microscopic Findings In the control case disorganization of the cytoskeletal elements, such as neurofilaments and neurotubulus was seen in some places. The most characteristic findings were separation of the myelin sheath, enlarge-
T. Yamaki etal.: Pathological Study of Diffuse Axonal Injury Patients Who Died Shortly After Impact
155
Fig. 1. Typical macroscopic findings (Case 2). There are haemorrhagic lesions in the corpus caltosum (long black arrow) and subcortical white matter (short black arrows), and also small lesions in the cerebral cortex (white arrows) (A). Small haemorrhagic lesions are seen in the dorsolateral quadrant, the periaquaeductal region, and also the ventrolateral region of the rostral brainstem in the same subject (B)
Fig. 2. Photomicrograph of slight axonal swelling in the corpus eallosum in Case 5. Bodian's silver impregnation (arrows) ( x 100) (A) and also toluidine blue stain (arrows) (x 160) (B)
ment o f the spaces between lamellae o f the myelin sheath, and detachment o f the a x o l e m m a f r o m the myelin sheath (Fig. 4). I n Case 1-7, the organization o f cytoskeletal elements was markedly impaired. We often f o u n d severe axonal swelling, in which the axoplasmic contents were extruded and the myelin sheath had separated and become very thin. The layers o f the myelin sheath were widely separated. However, no A R B s were demonstrated (Fig. 5). Similar electron microscopic findings were noted in Case 8 where the m a c r o - u n d microscopic findings seemed to be similar to Cases 1-7. Characteristic A R B s were also demonstrated in Case 8. M a n y
Fig. 3. Photomicrograph of typical axonal retraction bails in the corpus callosum in Case 8. Bodian's (x 100) (A) and toluidine blue stain (arrows) (x 160) (B)
axons o f this type possessed very swollen and clear axoplasm, and only a few cell organellae (Fig. 6).
Discussion M o s t reports in the literature on pathological findings o f closed head injury cases, w h o died shortly after the impact, are based on cases who survived m o r e than 12 hours 1' 2, 7, 12, 16 In this regard our material was different because, with one exception, our cases died within the first hour. Their macroscopical autopsy findings corresponded to D A I . In all cases the brain surface
156
T. Yamaki etal.: Pathological Study of Diffuse Axonal Injury Patients Who Died Shortly After Impact
Fig. 4. Electron/Photomicrograph of the corpus callosum in the control case. Separation of the myelin sheath (long arrow), enlargement of the spaces (asterisk) between myelin layers, and detachment of the axolemma from the myelin sheath (short arrows) are seen ( x 3,000)
and cut sections showed features like haemorrhages which are characteristically found in D A I 1, is. All cases showed subarachnoid haemorrhage (100%), 63% petechial haemorrhages in the subcortical white matter, 50% for the basal ganglia, 88% for the corpus callosum, and also 88% for the dorsolateral quadrant of the rostral brain stem. Cerebral contusions were located in the parasagittal regions of the frontal and parietal lobes in two cases. These contusions were so called gliding contusions. A d a m s et al. 3 reported that gliding contusions are strongly associated with the most severe type of DAI. H a e m o r r h a g e in the basal ganglia is also most often associated with severe type D A I 4. Recently the term of "Diffuse axonal injury" has been used not only as a histological entity but also as a clinical one 6, 10 and some confusion has arisen when the term " D A I " is used. We, however, believe that this
Fig. 5. Electron/Photomicrographs of the corpus callosum in Case 3. They show remarkable axonal swelling in which the axoplasmic contents are extruded (long arrow) and the myelin sheath layers are separated and very thin (short arrows). The spaces between myelin sheath layers are wide ( • 3,000) (A). In Case 5, the findings shown in photograph A) are much more prominent (B).
term should be used only as a histological entity, becaue diffuse damage to axons can only be proven by microscopic examination. The characteristic microscopic findings of D A I are haemorrhages which occur first in the perivascular region and then extend into the adjacent tissue, and numerous axonal retraction balls (ARBs) 1. However, ARBs are reported to be found only in cases who survive longer than 12 hours 5' 9, 12 Sahuquillo e t a I . ~6 also reported that 2 cases with a survival time of 10 hours did not have ARBs. On microscopic examination of our cases, we found small haemorrhagic lesions in areas as mentioned previously.
T. Yamaki et
al.:
Pathological Study of DiffuseAxonal Injury Patients Who Died Shortly After Impact
Fig. 6. Electron/PhotoreAcrographsof the corpus callosum in Case 8. There are similar findings to those demonstrated in Fig. 7 ( x 3,000) (A), but several types of axonal retraction balls are also seen (x 6,000) (B, C)
Axonal retraction balls were not found at all in Cases 1 to 7 despite the characteristic features of DAI noted macroscopically. In Case 8, typical ARBs were noted
157
with Bodian's silver impregnation and toluidine blue staining. The reason why ARBs were not found in Case 1 to 7 was probably because of the very short survival time. However, slight axonal swelling was observed in some of these subjects. Experimental studies of axonal injuries 8, 13, 15 have revealed that intra-axonal horseradish peroxidase (HRP) pooling, a maker for altered axons, occurs microscopically within one hour of the insult. With the progression of time, the HRP-laden segments enlarge and demonstrate various forms, and within 12-24 hours the intra-axonal swelling became a large conspicuous ball. Povlishock and Kontos 14 studied an experimental model of DAI and reported subtle focal abnormalities of axolemmal irregularities, microtubular clumping, and organelle pooling within one hour of trauma without using H R P or electron microscopy. The slight axonal swelling found in this study might be the same as that reported experimentally by these workers. An electron microscopic analysis of human DAI has not been reported previously. The reasons why electron microscopic studies of human DAI have not been done may include the following: 1) the diagnosis of DAI can be easily made by the characteristic findings on both macroscopic and/ or microscopic examination when patients survive for more than 12 hours, 2) the appropriate treatment of injured brains for electron microscopic examination is difficult shortly after death, 3) there are few autopsy cases who die almost immediately after injury. The most important factor is thought to be the second one, because rapid postmortem changes of the brain and fixation of the brain with formalin produce artifacts for electron microscopy. We ventured to study our cases by electron microscopy because on light microscopy in cases 1 to 7 there were only few findings, and because it is reported that only electron microscopic findings can prove the existence of axonal injury at a very early stage after the impact 12' 14 In the control case, some disorganization of the cytoskeletal elements was seen. These findings were thought to be artifacts due to postmortem changes of the brain tissue and/or inappropriate fixation for electron microscopy. Similar findings were also noted in the head injury cases, but the degree of destruction of the axons was much more severe. In Case 8, numerous ARBs were clearly seen on electron microscopic study. But we could not find any ARBs in Case 1 to 7. The characteristic electron microscopic findings in Cases 1
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T. Yamaki et al.: Pathological Study of Diffuse Axonal Injury Patients Who Died Shortly After Impact
to 7 were remarkable swelling of the axons, separation of lamellae of the myelin sheath and wide spaces between the myelin layers. In experimental studies TM a4, on low-intensity fluid percussion brain injury in the cat, the findings were similar to those in our human material, but the degree of cytoskeletal destruction was severer in our cases. These difference between the experimental models and our subjects may have been due not only to inappropriate treatment for electron microscopy but also to differences in the severity of the head injury. F r o m the results of our study, we think that there would be many cases of DAI among head injury patients who die within a very short interval after the impact. Electron microscopic examination of the human brain can verify the existence of DAI at a very early stage after injury.
References 1. Adams JH, Mitchell DE, Graham DI, Doyle D (1977) Diffuse brain damage of immediate impact type. Its relationship to "primary brain-stem damage" in head injury. Brain 100:489-502 2. Adams JH, Graham DI, Murray LS, Scott G (1982) Diffuse axonal injury due to nonmissile head injury in human: an analysis of 45 cases. Ann Neurol 12:557-563 3. Adams JH, Doyle D, Graham DI, Lawrence AE, McLellan DR (i 986 a) Gliding contusions in nonmissilehead injury in humans. Arch Pathol Lab Med 110:485-488 4. Adams JH, Doyle D, Graham DI, Lawrence AE, McLellan DR (1986 b) Deep intracerebral (basal ganglia) hematomas in fatal non-missile head injury in man. J Neurol Neurosurg Psychiatry 49:1039-1043 5. Adams JH, Doyle D, Ford I, Gennarelli TA, Graham DI, McLellan DR (1989) Diffuse axonal injury: definition, diagnosis and grading. Histopathology 15:49-59 6. Cordobes F, Lobato RD, Rivas JJ, Cabrera A, Sarabia M, Castro S, Cisneros C, Torres ID, Lamas E (1986) Post-traumatic
7. 8.
9.
10.
11.
12. 13.
14.
15. 16.
17.
18.
diffuse axonal brain injury. Analysis of 78 patients studied with computed tomography. Aeta Neuroehir (Wien) 81:27-35 Crompton MR (1971) Brain stem lesions due to closed head injury. Lancet 1:669-673 Erb DE, Povlishock JT (1988) Axonal damage in severe traumatic brain injury: an experimental study in eat. Acta Neuropathol (Berl) 76:347-358 Graham DI, Adams JH, Logan S, Gennarelli TA, Thibault L (1985) The distribution, nature and time course of diffuse axonal injury. Neuropathol Appl Neurobiol (Abstr) 11:319 Gennarelli TA (1984) Emergency department management of head injuries. Emergency Medicine Clinics of North America 2:749-760 Maxwell WL, Kansagra AM, Graham DI, Adams JH, Gennarelli TA (1988) Freeze-fracture studies of reactive myelinated nerve fibres after diffuse axonal injury. Aeta Neuropathol (Beil) 76:395-406 Pilz P (1983) Axonal injury in head injury. Acta Neuroehir (Wien) [Suppl] 32:119-123 Povlishock JT, Becker DP, Cheng CLY, Vaughan GW (1983) Axonal change in minor head injury. J Neuropathol Exp Neurol 42:225-242 Povlishock JT, Kontos HA (1985 a) Continuing axonal and vascular change following experimental brain trauma. Central Nervous System Trauma 2:285-298 Povlishock JT, Becker DP (1985 b) Fate of reactive axonal swelling induced by head injury. Lab Invest 52:540-552 Sahuquillo J, Vilata J, Lamarca J, Rubio E, Rodriguez-Pazos M, Salva JA (1989) Diffuse axonal injury after severe head tauma. A clinico-pathological study. Acta Neurochir (Wien) 101:149-158 Tomei G, Spagnoli D, Ducati A, Landi A, Villani R, Fumagalli G, Sala C, Gennarelli T (1990) Morphology and neurophysiology of focal axonal injury experimentally induced in the guinea pig optic nerve. Acta Neuropathol (Bed) 80:506-513 Zimmerman RA, Bilaniuk LT, Gennarelli TA (1978) Computed tomography of shearing injuries of the cerebral white matter. Radiology 27:393-396
Correspondence and Reprints: Tarumi Yamaki, M.D., Ph.D., Department ofNeurosurgery, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto, 602, Japan.
Acta Neurochir (Wien) (1992) 119:153-158
9 Springer-Verlag 1992 Printed m Austria
Pathological Study of Diffuse Axonal Injury Patients Who Died Shortly After Impact T. Yamaki ~, N. Murakami t, Y. Iwamoto 1, Y. Nakagawa 1, S. Ueda 1, Y. Irizawa 2, S. Komura 2, and T. Matsuura 3 Departments of 1Neurosurgery and 2Forensic Medicine, Kyoto Prefectural University of Medicine, and 3Department of Anatomy, Meiji College of Oriental Medicine, Kyoto, Japan
Summary
in 7 cases a n d in o n e o t h e r case 3 d a y s later. F o r
It is generally considered that axonal injury is apparent only on electron microscopy in the very early stage after a closed head injury. To clarify the pathological findings in head injury patients dying very shortly after the impact, we analyzed 8 fatal cases of diffuse axonal injury (DAI) who underwent medicolegal autopsy at the Department of Forensic Medicine of Kyoto Prefectural University of Medicine. Seven cases died within one hour after injury and another one case died 3 days after injury. We studied these cases macroscopically, microscopically, and electron microscopically. Macroscopically all cases showed the typical findings of diffuse axonal injury. Microscopical study of the cases who died within one hour revealed no characteristic findings of DAI such as appearance of retraction bails or microglia. On the other hand, in the case who died only 3 days after injury it showed the typical retraction balls. Electron microscopic study showed the remarkable destruction of cytoskeletal structure of axons in all cases. From our results, it is reasonable to speculate that DAI may be common among head injury patients who die very soon after the impact.
pathological examination, we studied the brains mac-
Keywords:Head injury; early death; diffuse axonal injury; pathological study.
Introduction Clinicopathological reports on human closed head i n j u r y are c o m m o n , b u t p a t h o l o g i c a l f i n d i n g s o f diffuse a x o n a l i n j u r y cases, w h e r e d e a t h o c c u r r e d i m m e d i a t e l y a f t e r t h e i m p a c t , h a v e r a r e l y b e e n r e p o r t e d . It is c o n s i d e r e d t h a t i m m e d i a t e l y f o l l o w i n g t h e initial insult, a x o n a l i n j u r y is o n l y a p p a r e n t b y e l e c t r o n m i c r o s c o p y . H o w e v e r , we c o u l d n o t f i n d a n y p a p e r s p u b l i s h e d o n e l e c t r o n m i c r o s c o p i c f i n d i n g s in the e a r l y stages o f h u m a n a x o n a l injury, a n d o n l y d a t a f r o m e x p e r i m e n t a l m o d e l s s e e m to be a v a i l a b l e 8' 9. 11, 17 T h e r e f o r e , w e s t u d i e d 8 f a t a l cases o f diffuse a x o n a l i n j u r y ( D A I ) . Among them, death occurred within one hour of injury
roscopically, microscopically, and electron microscopically.
Materials and Methods We studied 16 fatal cases of closed head injury and conducted medicolegal autopsies at the Department of Forensic Medicine of Kyoto Prefectural University of Medicine from 1988 to 1989. All of these subjects underwent a complete autopsy and it was confirmed that there were no lesions but those of the brain as the cause of death. Among them in 8 cases, the characteristic findings of DAI 15 were noted macroscopically. Those findings were diffuse subarachnoid haemorrhage, small haemorrhages in subcortical white matter, corpus callosum, basal ganglia, and brain stem. No findings of increased intracranial pressure such as brain swelling and/or large intracerebral haematoma were seen. The interval from death to autopsy of these cases ranged from 4 to 36 hours (Table 1). When the interval from death to autopsy was over 12 hours, the bodies were preserved in a refrigerator. After the autopsy, the whole brain was fixed in 10% formalin. Macroscopic forensic examination was done within one month after the fixation, while microscopic and electron microscopic examinations were done 1-2 years later. For the microscopic examination, representative tissue blocks were obtained from the frontal and temporal cortices, their subcortical white matter, the basal ganglia, the corpus callosum, and the pons. All the blocks were embedded in paraffin and 6 gm sections were cut and stained with haematoxylin-eosin (HE), Luxol fast blue, Bodian's silver impregnation, and toluidine blue. Electron microscopic examination of the corpus callosum and the dorsolateral quadrants of the rostral pons was also done. Specimens fixed in 10% formalin were cut into small pieces and fixed again with 2.5% glutaraldehyde and 1% osmium tetraoxide in 0.1 M phosphate buffer (pH 7.4). After fixation, the specimens dehydrated with graded series of acetone and embedded in Libeak 812. Ultrathin sections were cut on a Reichert Ultracut, stained with uranyl
T. Yamaki etal.: Pathological Study of Diffuse Axonal Injury Patients W h o Died Shortly After Impact
154
Table 1. Clinical Profile of the Autopsy Cases No. Age
Sex
Cause of head injury
Impact site
Skull facture
Autopsy findings (Brain surface)
Intervals from impacts to death
Intervals from death to autopsy
1 2 3 4 5 6 7 8
M M M M M F F M
traffic accident unknown traffic accident traffic accident traffic accident traffic accident traffic accident fall
Rt. Rt. Lt. Rt. Rt. Lt. Rt. Lt.
+ + + + + + + -
SAH, CC SAH SAH, A S H SAH, CC SAH SAH SAH SAH
1h 1h 1h 1h 1h 1h 1h 3d
4h 36 h 10h 32h 11 h 15 h 19 h 24 h
5 18 41 47 50 65 78 19
temporal temporal occipital frontal temporal temporal frontal occipital
S A H subarachnoid haemorrhage, C C cerebral contusion, A S H acute subdural haematoma.
Table 2. Sites of Haemorrhagic Lesions. Findings from the examination of brain slices Case no.
Cortex
Subcortical white matter
I
+
+
+
+
+
2 3 4 5 6 7 8
+ + -. --3/8
+ + + +
-+ + +
+ + + +
-4/8
+ + 7/8
+ + + + + -+ 7/8
.
.
Basal ganglia
Corpus callosum
.
-5/8
Brain stem
acetate and lead nitrate, and examined using a J E D L S-100 electron microscope. Artifacts often affect electron microscopy findings, so, as a control we also studied the corpus callosum and dorsal pons of a 67-year-old w o m a n who died suddenly due to myocardial infarction, a n d underwent a medicolegal autopsy in 1988, which was prepared in the same m a n n e r as the head injury cases.
Results
Clinical Profile of the Autopsy Cases The ages of the cases ranged from 5 to 78 years. Six of the subjects were males and 2 were females. Six cases were injured in traffic accidents, 1 by a fall and 1 by some unknown cause. Four cases were transferred to hospital by an ambulance but 3 of them were "dead on arrival" except for Case 8. Another 4 cases were directly transferred to the Department of Forensic Medicine (Table 1).
Autopsy Findings of the Head Four out of 8 cases had an impact to the temporal region, 2 to the frontal region, and one to the occipital
region. Skull fractures were noted in 7 cases except for Case 8. Intracranial haematoma (a thin acute subdural haematoma) was seen in only one case out of 8 cases. The brain surface showed traumatic subarachnoid haemorrhage in all cases. The sites of intracerebral haemorrhage on cut sections of the brain are shown in Table 2. Cortical haemorrhage was found in 3/8 cases (38%), 5 cases (63%) had it in the subcortical white matter, 4 cases (50%) had basal ganglia haemorrhage, 7 cases (88%) hat it in the corpus callosum, and 7 cases (88%) had brain stem haemorrhage. All of the haemorrhages were small or petechial (Fig. 1). Cerebral contusion was noted in 2 cases (25%). These contusions were small and located on the parasagittal portion of the frontal and parietal lobes.
Microscopic Findings On microscopy, only the haemorrhagic lesions were seen with H E staining. In the cases who died within one hour (cases 1-7) no remarkable axonal changes such as axonal retraction balls (ARBs) were noted in the subcortical white matter, corpus callosum, or dorsolateral quadrants of the rostral pons, in sections stained with both Bodian's silver impregnation and toluidine blue. However, slight reactive axonal swelling was seen in 4 out of 7 cases (57%) (Fig. 2). In Case 8 who survived for 3 days, typical ARBs were noted (Fig. 3).
Electron Microscopic Findings In the control case disorganization of the cytoskeletal elements, such as neurofilaments and neurotubulus was seen in some places. The most characteristic findings were separation of the myelin sheath, enlarge-
T. Yamaki etal.: Pathological Study of Diffuse Axonal Injury Patients Who Died Shortly After Impact
155
Fig. 1. Typical macroscopic findings (Case 2). There are haemorrhagic lesions in the corpus caltosum (long black arrow) and subcortical white matter (short black arrows), and also small lesions in the cerebral cortex (white arrows) (A). Small haemorrhagic lesions are seen in the dorsolateral quadrant, the periaquaeductal region, and also the ventrolateral region of the rostral brainstem in the same subject (B)
Fig. 2. Photomicrograph of slight axonal swelling in the corpus eallosum in Case 5. Bodian's silver impregnation (arrows) ( x 100) (A) and also toluidine blue stain (arrows) (x 160) (B)
ment o f the spaces between lamellae o f the myelin sheath, and detachment o f the a x o l e m m a f r o m the myelin sheath (Fig. 4). I n Case 1-7, the organization o f cytoskeletal elements was markedly impaired. We often f o u n d severe axonal swelling, in which the axoplasmic contents were extruded and the myelin sheath had separated and become very thin. The layers o f the myelin sheath were widely separated. However, no A R B s were demonstrated (Fig. 5). Similar electron microscopic findings were noted in Case 8 where the m a c r o - u n d microscopic findings seemed to be similar to Cases 1-7. Characteristic A R B s were also demonstrated in Case 8. M a n y
Fig. 3. Photomicrograph of typical axonal retraction bails in the corpus callosum in Case 8. Bodian's (x 100) (A) and toluidine blue stain (arrows) (x 160) (B)
axons o f this type possessed very swollen and clear axoplasm, and only a few cell organellae (Fig. 6).
Discussion M o s t reports in the literature on pathological findings o f closed head injury cases, w h o died shortly after the impact, are based on cases who survived m o r e than 12 hours 1' 2, 7, 12, 16 In this regard our material was different because, with one exception, our cases died within the first hour. Their macroscopical autopsy findings corresponded to D A I . In all cases the brain surface
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T. Yamaki etal.: Pathological Study of Diffuse Axonal Injury Patients Who Died Shortly After Impact
Fig. 4. Electron/Photomicrograph of the corpus callosum in the control case. Separation of the myelin sheath (long arrow), enlargement of the spaces (asterisk) between myelin layers, and detachment of the axolemma from the myelin sheath (short arrows) are seen ( x 3,000)
and cut sections showed features like haemorrhages which are characteristically found in D A I 1, is. All cases showed subarachnoid haemorrhage (100%), 63% petechial haemorrhages in the subcortical white matter, 50% for the basal ganglia, 88% for the corpus callosum, and also 88% for the dorsolateral quadrant of the rostral brain stem. Cerebral contusions were located in the parasagittal regions of the frontal and parietal lobes in two cases. These contusions were so called gliding contusions. A d a m s et al. 3 reported that gliding contusions are strongly associated with the most severe type of DAI. H a e m o r r h a g e in the basal ganglia is also most often associated with severe type D A I 4. Recently the term of "Diffuse axonal injury" has been used not only as a histological entity but also as a clinical one 6, 10 and some confusion has arisen when the term " D A I " is used. We, however, believe that this
Fig. 5. Electron/Photomicrographs of the corpus callosum in Case 3. They show remarkable axonal swelling in which the axoplasmic contents are extruded (long arrow) and the myelin sheath layers are separated and very thin (short arrows). The spaces between myelin sheath layers are wide ( • 3,000) (A). In Case 5, the findings shown in photograph A) are much more prominent (B).
term should be used only as a histological entity, becaue diffuse damage to axons can only be proven by microscopic examination. The characteristic microscopic findings of D A I are haemorrhages which occur first in the perivascular region and then extend into the adjacent tissue, and numerous axonal retraction balls (ARBs) 1. However, ARBs are reported to be found only in cases who survive longer than 12 hours 5' 9, 12 Sahuquillo e t a I . ~6 also reported that 2 cases with a survival time of 10 hours did not have ARBs. On microscopic examination of our cases, we found small haemorrhagic lesions in areas as mentioned previously.
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al.:
Pathological Study of DiffuseAxonal Injury Patients Who Died Shortly After Impact
Fig. 6. Electron/PhotoreAcrographsof the corpus callosum in Case 8. There are similar findings to those demonstrated in Fig. 7 ( x 3,000) (A), but several types of axonal retraction balls are also seen (x 6,000) (B, C)
Axonal retraction balls were not found at all in Cases 1 to 7 despite the characteristic features of DAI noted macroscopically. In Case 8, typical ARBs were noted
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with Bodian's silver impregnation and toluidine blue staining. The reason why ARBs were not found in Case 1 to 7 was probably because of the very short survival time. However, slight axonal swelling was observed in some of these subjects. Experimental studies of axonal injuries 8, 13, 15 have revealed that intra-axonal horseradish peroxidase (HRP) pooling, a maker for altered axons, occurs microscopically within one hour of the insult. With the progression of time, the HRP-laden segments enlarge and demonstrate various forms, and within 12-24 hours the intra-axonal swelling became a large conspicuous ball. Povlishock and Kontos 14 studied an experimental model of DAI and reported subtle focal abnormalities of axolemmal irregularities, microtubular clumping, and organelle pooling within one hour of trauma without using H R P or electron microscopy. The slight axonal swelling found in this study might be the same as that reported experimentally by these workers. An electron microscopic analysis of human DAI has not been reported previously. The reasons why electron microscopic studies of human DAI have not been done may include the following: 1) the diagnosis of DAI can be easily made by the characteristic findings on both macroscopic and/ or microscopic examination when patients survive for more than 12 hours, 2) the appropriate treatment of injured brains for electron microscopic examination is difficult shortly after death, 3) there are few autopsy cases who die almost immediately after injury. The most important factor is thought to be the second one, because rapid postmortem changes of the brain and fixation of the brain with formalin produce artifacts for electron microscopy. We ventured to study our cases by electron microscopy because on light microscopy in cases 1 to 7 there were only few findings, and because it is reported that only electron microscopic findings can prove the existence of axonal injury at a very early stage after the impact 12' 14 In the control case, some disorganization of the cytoskeletal elements was seen. These findings were thought to be artifacts due to postmortem changes of the brain tissue and/or inappropriate fixation for electron microscopy. Similar findings were also noted in the head injury cases, but the degree of destruction of the axons was much more severe. In Case 8, numerous ARBs were clearly seen on electron microscopic study. But we could not find any ARBs in Case 1 to 7. The characteristic electron microscopic findings in Cases 1
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to 7 were remarkable swelling of the axons, separation of lamellae of the myelin sheath and wide spaces between the myelin layers. In experimental studies TM a4, on low-intensity fluid percussion brain injury in the cat, the findings were similar to those in our human material, but the degree of cytoskeletal destruction was severer in our cases. These difference between the experimental models and our subjects may have been due not only to inappropriate treatment for electron microscopy but also to differences in the severity of the head injury. F r o m the results of our study, we think that there would be many cases of DAI among head injury patients who die within a very short interval after the impact. Electron microscopic examination of the human brain can verify the existence of DAI at a very early stage after injury.
References 1. Adams JH, Mitchell DE, Graham DI, Doyle D (1977) Diffuse brain damage of immediate impact type. Its relationship to "primary brain-stem damage" in head injury. Brain 100:489-502 2. Adams JH, Graham DI, Murray LS, Scott G (1982) Diffuse axonal injury due to nonmissile head injury in human: an analysis of 45 cases. Ann Neurol 12:557-563 3. Adams JH, Doyle D, Graham DI, Lawrence AE, McLellan DR (i 986 a) Gliding contusions in nonmissilehead injury in humans. Arch Pathol Lab Med 110:485-488 4. Adams JH, Doyle D, Graham DI, Lawrence AE, McLellan DR (1986 b) Deep intracerebral (basal ganglia) hematomas in fatal non-missile head injury in man. J Neurol Neurosurg Psychiatry 49:1039-1043 5. Adams JH, Doyle D, Ford I, Gennarelli TA, Graham DI, McLellan DR (1989) Diffuse axonal injury: definition, diagnosis and grading. Histopathology 15:49-59 6. Cordobes F, Lobato RD, Rivas JJ, Cabrera A, Sarabia M, Castro S, Cisneros C, Torres ID, Lamas E (1986) Post-traumatic
7. 8.
9.
10.
11.
12. 13.
14.
15. 16.
17.
18.
diffuse axonal brain injury. Analysis of 78 patients studied with computed tomography. Aeta Neuroehir (Wien) 81:27-35 Crompton MR (1971) Brain stem lesions due to closed head injury. Lancet 1:669-673 Erb DE, Povlishock JT (1988) Axonal damage in severe traumatic brain injury: an experimental study in eat. Acta Neuropathol (Berl) 76:347-358 Graham DI, Adams JH, Logan S, Gennarelli TA, Thibault L (1985) The distribution, nature and time course of diffuse axonal injury. Neuropathol Appl Neurobiol (Abstr) 11:319 Gennarelli TA (1984) Emergency department management of head injuries. Emergency Medicine Clinics of North America 2:749-760 Maxwell WL, Kansagra AM, Graham DI, Adams JH, Gennarelli TA (1988) Freeze-fracture studies of reactive myelinated nerve fibres after diffuse axonal injury. Aeta Neuropathol (Beil) 76:395-406 Pilz P (1983) Axonal injury in head injury. Acta Neuroehir (Wien) [Suppl] 32:119-123 Povlishock JT, Becker DP, Cheng CLY, Vaughan GW (1983) Axonal change in minor head injury. J Neuropathol Exp Neurol 42:225-242 Povlishock JT, Kontos HA (1985 a) Continuing axonal and vascular change following experimental brain trauma. Central Nervous System Trauma 2:285-298 Povlishock JT, Becker DP (1985 b) Fate of reactive axonal swelling induced by head injury. Lab Invest 52:540-552 Sahuquillo J, Vilata J, Lamarca J, Rubio E, Rodriguez-Pazos M, Salva JA (1989) Diffuse axonal injury after severe head tauma. A clinico-pathological study. Acta Neurochir (Wien) 101:149-158 Tomei G, Spagnoli D, Ducati A, Landi A, Villani R, Fumagalli G, Sala C, Gennarelli T (1990) Morphology and neurophysiology of focal axonal injury experimentally induced in the guinea pig optic nerve. Acta Neuropathol (Bed) 80:506-513 Zimmerman RA, Bilaniuk LT, Gennarelli TA (1978) Computed tomography of shearing injuries of the cerebral white matter. Radiology 27:393-396
Correspondence and Reprints: Tarumi Yamaki, M.D., Ph.D., Department ofNeurosurgery, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto, 602, Japan.
