Methods in Forensic Neuropathology

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Methods in Forensic Neuropathology 5

5.1 Macroscopic Examination 83 5.1.1 Principles of Brain Autopsy 83 5.1.1.1 Biosafety 83

5.1.1.2 Preserved Native Material 83 5.1.1.3 Documentation 84 5.1.2 Brain 84

5.1.2.1 Fixation Procedures 84 5.1.2.2 Examination during Autopsy 85 5.1.2.3 Sectioning

of the Formalin-Fixed Brain 85 5.1.2.4 Block Selection for Microscopy 86 5.1.2.5 Paraffin Embedding 88

5.1.2.6 Special Problems 88 5.1.3 Spinal Cord 89 5.1.4 Peripheral Nerves 89 5.2 Microscopic Examination 90 5.3 Postmortem Imaging 90

Bibliography 92

References 92

5.1

Macroscopic Examination

5.1.1

Principles of Brain Autopsy

5.1.1.1 Biosafety

Autopsy of the brain must begin by making certain that precautions have been taken to ensure biosafety (Nolte et al. 2002). Contact with droplets and/or di- rect cutaneous inoculation (percutaneous injury), even after the brain has been fixed in formalin, en- tails a risk of autopsy-transmitted infection. The overrepresentation of intravenous drug users among the forensic autopsy population means that the prev- alence of hepatitis B virus (HBV), hepatitis C virus

(HCV), and human immunodeficiency virus (HIV) is disproportionately high. Blood infected with HIV has a 0.3% risk of transmission per exposure, blood infected with HBV a 30% risk (Shapiro 1995), and HCV infected blood a 1.8 to 10% risk (Centers for Disease Control and Prevention 1998). Bodies infected with HIV are infectious for at least 2 weeks postmortem; after being dried and exposed to an ambi- ent environment, HBV in human plasma remained infectious for 1 week (Bond et al. 1981).

Because relatively high risk is associated with autopsies on victims of viral hemorrhagic fever, a careful assessment of the risk and benefits should be made before autopsy. Autopsies on victims of Hanta- an virus infections are less hazardous than those on victims of other viral hemorrhagic fever infections.

Creutzfeldt-Jakob disease (CJD), bovine spongi- form encephalopathy (BSE) and other spongiform encephalopathies can be acquired by percutaneous exposure during autopsy. Infectious isoforms of host-membrane sialoglycoproteins known as prions cause transmissible dementias (Johnson and Gibbs 1998). Formalin does not inactivate prions and they retain their ability to be transmitted in paraffin blocks (Brown et al. 1986). Two histology technicians contracted CJD while working in neuropathology laboratories (Miller 1988). Guidelines for safe performance of autopsies, processing and handling tissues, decontaminating work surfaces and instruments are given in Table 5.1 (cf. Schulze-Schaeffer et al. 1998; Nolte et al. 2002). A contamination has to be performed with 1 N NaOH followed by 1 N HCl and rinsed water. Procedures for brain cutting must be carefully performed according to recommendation in Table 5.2.

5.1.1.2

Preserved Native Material

Specimens or liquids obtained at autopsy should be preserved for further investigations on native specimens. Exactly what tissues and how much are preserved depend on the nature of the problem to be solved. Specimens should be examined immediately, frozen or subjected to specific treatment (fixation with special agents). Table 5.3 lists a number of

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forms of preservation that should be routinely applied in every case.

5.1.1.3

Documentation

Photographic documentation of pathological chang- es is an established part of macroscopic and microscopic examination in forensic neuropathology. As a means of documentation such pictures are worth many words of dry description. A scaled tape or ruler with clearly visible graduations may be placed within the photographic field to provide information on the size of the pathological finding, especially if it is a laceration, hemorrhage, or cavitation. The camera should be aimed at right angles to the surface of the slide or specimen.

Radiography is especially indicated before autop- sy of victims of gunshot or of suspected child abuse.

X-ray, CCT, and/or MRI can provide additional documentation for reconstruction as well as information essential to planning the autopsy, especially in cases involving embedded projectiles or old fractures (see pp. 90 ff).

5.1.2 Brain

In the field of forensic neuropathology the same techniques are to be applied as in clinical neuropathology. Therefore, textbooks of neuropathologic techniques are recommended, e.g., Dawson et al.

(2003). The following short methodical description

gives an introduction and general information on the applied techniques.

5.1.2.1

Fixation Procedures

The brain must not be examined fresh, or roughly 50% of lesions will be missed, compromising further examination, referral, and ultimately the verity of evidence presented in court. Fixation in 10% buffered formalin prior to routine examination by sectioning is thus essential. To be of good quality, fixa- tion of the brain in formalin must meet the following requirements:

▬ The formalin is neutral and buffered

▬ The quantity is ample (10 times the volume of the brain) and at the right concentration (not less than 10% concentration)

▬ The fixation time is at least 2 weeks, but does not exceed 4 weeks (formalin becomes acidic at fixation times over 4 weeks)

Two techniques are used for brain fixation: the immersion technique and the perfusion technique. In the immersion technique the brain is removed in its

Table 5.1. Procedures for decontaminating instruments and tables in cases of suspected Creutzfeldt-Jakob disease or bovine spongiform encephalopathy. Source:

Nolte et al. 2002

1. Instruments and saw blades are placed into a large stainless steel dish, soaked for 1 h in 2 N sodium hydroxide or 2 h in 1 N sodium hydroxide, and then rinsed thoroughly in water before autoclaving at 134°C [gravity displacement steam autoclaving for 1 h; porous load steam autoclaving for one 18-min cy- cle at 200 kPa (2 atm) or six 3-min cycles at 200 kPa]

2. The Stryker saw is cleaned by repeated wetting with 2 N sodium hydroxide solution over a 1-h period 3. Any suspected areas of contamination of the autopsy

table or room are decontaminated by repeated wetting over 1 h with 2 N sodium hydroxide

Table 5.2. Procedures for tissue preparation and cutting procedures in cases of suspected Creutzfeldt-Jakob disease or bovine spongiform encephalopathy. Source:

Nolte et al. 2002

1. Formaldehyde fixation: the brain is examined and cut on the table covered with an absorbent pad with an impermeable backing

2. Samples for histology are placed in cassettes labeled

“CJD precautions.” Blocks of formalin-fixed tissue can be placed in 96% absolute formic acid for 60 min, followed by fresh 10% neutral buffered formalin solution for at least 48 h. Embedding in paraffin as usual 3. All instruments and surfaces are decontaminated 4. Routine staining:

a. Slides are processed by hand

b. Reagents are prepared in 100-ml disposable specimen cups

c. After placing the coverslip on, slides are decontaminated by soaking them for 1 h in 2 N NaOH d. Slides are labeled “Infectious-CJD”

5. Tissue remnants, cutting debris, and contaminated formaldehyde solution should be discarded within a plastic container as infectious hospital waste for eventual incineration

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natural state prior to any fixation. This has the advantage that untreated tissue and cerebrospinal fluid (CSF) can be obtained. The drawback is the risk of artificial wounding to the brain during removal and a fixation time lasting at least 14 days. There are two ways to avoid the creation of artifacts during fixation of the brain in formalin: table salt can be added to the formalin, which allows the brain to float freely in the solution; or, the brain can be suspended from a string tied to the basilar artery.

The perfusion technique achieves optimal fixa- tion of the brain in situ within 2−3 h. Formalin is in- fused into the brain via carotid artery stumps. Per- fusion is especially advisable if autolysis of the brain has already begun but not advanced far enough to prevent fluid flow. This technique minimizes the risk of artificial lesions being created during removal from the skull. Further macroscopic examination can be performed immediately thereafter.

5.1.2.2

Examination during Autopsy

Before the brain is removed the dura mater should be inspected for lacerations, punctures, external and subdural hemorrhage, thrombosis of the venous si- nus, surgical wounds, etc. External examination of the brain begins at autopsy and provides information regarding subarachnoid hemorrhages, tumors, inflammatory processes, etc. If a basilar aneurysm has ruptured, the brain requires further preparation before fixation since it is easier to remove sub- arachnoidal blood and expose the aneurysm in the unfixed brain.

The CSF should also be removed prior to fixation of the brain. It can be drawn off either from the large

basal cisterna during removal of the brain, or by lumbar puncture through the intervertebral disks in the area of the lumbar spine on the ventral side after removal of the internal organs from the abdominal cavity. For toxicological analysis native brain tissue should be removed from a region with no pathological changes, from the occipital lobe for example.

5.1.2.3

Sectioning of the Formalin-Fixed Brain

When obtaining brain sections a uniform procedure should be used to allow consistent written and/or photographic documentation of any external and internal lesions. The formalin-fixed brain should first be weighed, followed by a detailed description of the outer structure of the dura mater, the large external vessels, leptomeninges convolutions and any signs of brain swelling (herniation of the cerebellum and/or hippocampus, flattening of the gyri, etc.). The pro- cedure should proceed as outlined by, for example, Vonsattel et al. (1995).

Parallel sections are then cut through the cerebral hemispheres once the cerebellum and brain stem have been excised cutting through the cerebral peduncles (Fig. 5.1a). Sections of the cerebellum and brain stem (Fig. 5.1b−d) can also be obtained along the frontal plane, the cerebellum and brain stem thus being cut together with the cerebellar peduncles. This demonstrates the fourth ventricle to best advantage, useful in cases of subarachnoid hemorrhage, for example. Alternatively, sections can be cut longitudi- nally through the vermis of the cerebellum up to the fourth ventricle; the cerebellar peduncles are then excised to isolate both cerebellar hemispheres near the brain stem. Frontal incisions are then made through

Table 5.3. Types of specimens, handling of body fluids and tissues for different types of examination

Specimen Handling Type of examination

Cerebrospinal fluid Native/freezing Electrocytes

Toxic agents

Native Infectious agents

Brain tissue Native/freezing Toxic agents

Neurochemical diagnosis (lipidoses)

Native Infectious agents

Native/freezing Histochemistry

Formalin or other fixatives Immunohistochemistry Molecular biology

Glutaraldehyde Electron microscopy

Native/freezing Molecular biology

Formalin Histology (routine)

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the brain stem, medulla oblongata, and superior cervical cord. Parasagittal sections are then cut through the cerebellar hemispheres parallel to the cut surface of the cerebellar vermis. This method demonstrates alcoholic superior vermal atrophy to best advantage.

The method of cutting the cerebellum should thus be tailored to the case. In degenerative or alcoholic disease of the cerebellum, cutting in the parasagittal plane will best demonstrate the lesions, whereas in conditions dilating or impinging on the fourth ventricle, cutting the cerebellum attached to the brain stem will best preserve the relations that need to be seen.

The sections of the cerebrum can be cut in the frontal (coronal) plane, corresponding to the classi- cal procedure of clinical neuropathology (Fig. 5.2).

Or they can be cut parallel to the hat line (Fig. 5.3), the cut surfaces thus correlating with clinical im-

ages of axial CCT or MRT (Matsui and Hirano 1978).

A large knife held at right angles to the base of the brain or the frontal pole can be used to cut sections.

The sections should be no more than 1 cm thick. This can be achieved freehand, using a pair of specially designed brain angles or even using pipettes across which the knife is rolled. These techniques will obviate the appearance of knife lines across the surfaces of the cut sections, aiding the presentation appearance of photographic evidence.

5.1.2.4

Block Selection for Microscopy

If gross lesions are found during sectioning of the brain, selecting blocks becomes relatively easy. If a tumor is involved, a correct impression of the cytol- ogy and architecture can usually be obtained from

Fig. 5.1a−d. After inspection and documentation of the brain surface the cerebellum and brain stem have been excised cutting through the cerebral peduncles (a), cutting through the cerebel-

lar vermis (b), separating both the cerebellar hemispheres (c) and making parasaggital incisions through the brain stem as well as parasagittal sections through the cerebellum (d)

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blocks obtained from a well-defined border of the le- sion. In the case of cortical contusion injuries or vas- cular lesions, it is necessary to take a sizeable block extending the entire width of the glass slide and including at least one edge of the contusion injury and adjacent cortex. If there has been hemorrhaging in the brain, the presence of possible degenerative or inflammatory changes in the vessels can only be determined if the blocks originate from the ganglionic or medial side of the lesion and from the contralateral portion of the brain. The hemorrhagic margins in such cases should be scrutinized for small, easily overlooked anomalous vessels.

Proper selection of blocks is more difficult in brains with no apparent gross lesions. The clinical record can sometimes provide information on the topography of the lesions (Courville 1964). If the deceased has suffered from mental deterioration, cortical-subcortical blocks should be obtained from the midfrontal area; if there was known paralysis, blocks should be taken from the contralateral motor cortex or the motor pathways; blocks from patients with epilepsy should include the motor cortex, uncus or hippocampus; if there was documented visual fail- ure, tissue from the calcarine cortex, optic chiasm,

and optic tract must be examined; in cases of ataxia, blocks from the cerebellar cortex (including dentate nucleus), medulla (including olivary nucleus), and spinal cord are needed; if the victim has died of sud- den death in a febrile state, blocks should be made of the upper spinal cord, pons, medulla, thalamus, and midbrain.

If the clinical record indicates no specific area of the brain and no gross lesions are present, multiple blocks may be necessary. They can be of various sizes and shapes (triangular, rectangular, elongated, square, etc.) and should enable identification of the topography on the histological slide, as described by Courville (1964). Notches can be made on the block to distinguish the right and left hemispheres. Our own experience with routine cases has shown that the following areas of the brain must be preserved for microscopic examination (see Figs. 5.2, 5.3):

1. Frontal cortex (first and second frontal gyrus including white matter)

2. Lenticular nucleus 3. Thalamic nuclei

4. and 5. Hippocampus (right and left) 6. Corpus callosum (including caudate nuclei)

Fig. 5.2a–f. Frontal sections through the cerebrum and block selection. The sections were cut parallel to the frontal plane and marked blocks are selected for histological examination

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7. Cerebellum (including cortex and dentate nucleus)

8. Pons 9. Medulla

If brain tissue from a region is embedded in paraffin, tissue from the same area should also be preserved

in formalin. Frozen sections may subsequently be needed for metallic methods or special stains.

5.1.2.5

Paraffin Embedding

The paraffin-embedding procedure must avoid too rapid a dehydration or overheating of paraffin, which can induce shrinkage of neuropil and cells, artefac- tually giving rise to vast empty spaces around blood vessels and neurons. We recommend the following paraffin-embedding procedure:

▬ 6 h in ethanol (70%), +40°C

▬ 4 h each in ethanol (80%, 90%, 3×100%), +40°C

▬ 2 h each in xylol, 3×, +40°C

▬ 1 h each in paraffin, 3×, +60°C

▬ 3 h in paraffin, +60°C 5.1.2.6

Special Problems

Identification of CNS Tissue. Forensic practice requires not only the personal identification of tissue samples, for which DNA techniques are indicated, but also the identification of specific types of tissue, whether, for example, a particular piece of tissue de- rives from the brain or some other organ. For this purpose morphological criteria (Oehmichen 1984;

Oehmichen et al. 1984) can be applied as well as immunohistochemical methods using antibodies specific for the brain, or specific for glial or neuronal epitopes.

Kimura et al. (1995) showed that the ratio between the non-muscle myosin heavy chain isoforms M II A and M II B differs from tissue to tissue. They devel- oped a highly sensitive ELISA to quantify each heavy chain isoform of myosin and establish the traits of different types of brain tissue.

Brain Tissue of Mummies. Only a few investigators have examined the brain tissue of mummies. Gersz- ten and Martínez (1995) investigated the brains of 15 naturally mummified humans dating from 1,000 BC to 1,500 AD and excavated from the deserts of north- ern Chile. Macroscopically the cerebral hemispheres, cerebellum, dura mater, and spinal cord of several cases were found to be relatively well preserved. Five mummies exhibited signs of intracranial disease, three of external injury. One mummy had evidence of subarachnoid, one of intracerebral hemorrhage.

The brain parenchyma exhibited on light microscopy vascular structures against an eosinophilic staining background but few cellular elements. The architecture of the dura mater consisted, as is normal, of collagen fibrils.

Fig. 5.3a–c. Sections cut parallel to the hat line. The marked blocks are selected for histological investigation

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5.1.3 Spinal Cord

The spinal cord including the dura mater is carefully removed from the spinal canal according to recom- mendations made elsewhere (Hill and Anderson 1988). If a Rokitansky evisceration has been done as part of the general autopsy, then the spinal cord can easily be removed anteriorly by cutting the laminae with a Stryker saw. Posterior removal of the spinal cord is more laborious, involving posterior laminec- tomy. Posterior removal is essential, however, in cases where the cervical spinal cord must be seen in situ and relationships determined, such as Arnold−Chiari malformation or the presence of prior surgery in the cervical region. Whether removed by the anterior or posterior route, the cord should be preserved in a hanging position in such a manner that preserves the dura mater.

Macroscopic examination should begin with inspection of the outer and inner surfaces of the dura mater, followed by examination of the leptomeninges and leptomeningeal vessels. The spinal cord is then cut into pieces and cross sections scrutinized.

Blocks should be made from the various segments of the spinal cord.

The cervical spine including the occipitocervical junction must be removed in cases of mechanical brain injury (MBI) or any case involving mechanical injury of the neck, e.g., during strangulation, to ac- quire supplementary biomechanical information regarding the cause of death. Berzlanovich et al. (1998) have described the technique for dissecting the cervical vertebral column. The removed sample is fixed in 4% formalin for at least 14 days. After fixation, a circular saw is used to cut the cord in the sagittal direction along the midline. Cartilaginous and bony tissues are evaluated for fractures, tumor, hemor- rhages, necrosis, etc. The spinal canal is inspected for cord compression and/or stenosis. For evaluation of dural hemorrhages, the spinal cord and spinal dura are removed. The isolated spinal cord is cut into multiple cross sections and the cut surfaces examined for spinal hemorrhage, necrosis, cysts, and/or inflammation.

Preparation and documentation of the cervical spine must focus on the following structures:

▬ The vertebral body (degenerative alteration?;

fracture?; luxation?; presence and extent of he- matopoiesis?)

▬ The intervertebral disc (laceration?; hemorrhage?; complete or partial transection?)

▬ The spinal canal (cord compression?; stenosis?)

▬ The spinal dura (extradural or subdural hemorrhage?)

▬ The spinal cord (softening?; hemorrhage?; scarring?)

If macroscopic examination discloses pathological findings, soft tissues, bones, and vessels must be removed for embedding and histological examination.

In cases such as unexplained cerebellar infarction, where vertebral dissection is suspected (Auer et al.

1994), particular attention must be paid to the vertebral arteries for signs of trauma and/or thrombosis. After fixation in formalin, the vertebral arteries on both sides must be removed by bilateral excision.

Longitudinal and cross sections from the spinal cord are embedded and examined microscopically, especially for signs of axonal injury or a cell reaction.

5.1.4

Peripheral Nerves

Peripheral nerves need only be inspected if disease or injury is suspected. Material is garnered from peripheral nerves that could be of clinical or forensic interest.

Diagnosis of mechanical, poisoning or ischemic insults depends in large part on the morphological examination of peripheral nerves. The morphology remains optimally preserved if the tissue is fixed immediately in glutaraldehyde and embedded in plastic. Success often depends on early fixation of the nerve. Semithin and/or ultrathin sections are cut. In routine forensic neuropathological practice, any one of a number of stains and/or antibody reactions are used on 5-µm sections of paraffin-embedded tissue, among them the following:

Table 5.4. Histologic demonstration of different tissue structures in peripheral nerves by different staining techniques

Axonal injury Silver stain β-APP expression Collagen fibers Trichrome stain Macrophage activation CD 68 expression Myelin injury Klüver−Barrera stain

Basic myelin protein expression Neuronal injury H&E stain

Nissl stain Oligodendrocytes/

Schwann cells

Carbonic anhydrase II expression

Most institutes of forensic pathology are as ill equipped for the investigation of peripheral nerves

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as they are for investigation of muscles. Forensic pa- thologists also often lack the necessary experience for their proper examination. Difficult cases, therefore, must be referred to clinical neuropathological colleagues.

5.2

Microscopic Examination

As a rule, the thickness of paraffin sections should not exceed 5 µm. The most common stain for paraffin sections is hematoxylin and eosin stain (H&E).

Blue hematoxylin, a base, stains acidic cell structures such as nuclei, nucleoli, and Nissl bodies, while the acidic eosin stains basic components, mainly proteins, of cytoplasm, red blood cells, glial cell fibers, and collagenous fibers.

A number of special stains are used in classic neuroanatomy and neuropathology, which will be only briefly listed here together with their target tissue (for an overview see Luna 1960; Rális et al. 1973;

DeLellis 1988; Böck 1989):

Table 5.5. Routine techniques which demonstrate different structures of the CNS

Neurons: cresyl violet stain (Feigin and Cravioto 1961) Myelin: Luxol Fast Blue

Neurons and myelin: cresyl violet/Luxol Fast Blue (Klüver and Barrera 1953)

Axons: silver techniques (Bielschowsky, − see Hirano and Zimmermann 1962)

Glial fibers: silver technique (Holzer 1921) Astrocytes: silver technique (Cajal 1913)

Microglia: silver technique (Naoumenko and Feigin 1963)

Oligodendrocyte: silver technique (Conn and Darow 1943)

Polymorphonuclear (neutrophilic) leukocytes:

histochemistry – naphtol-AS-D chloracetate esterase (N-AS-DCIAE – see Böck 1989)

Reticulin fibers: silver technique (Gomori 1937)

In recent years numerous antibodies have been de- veloped for immunohistochemical techniques. These antibodies are capable of highly specific staining of cells, fibers, etc. The entire spectrum will not be

presented here, only antibodies known to be selec- tive for specific cells and fibers of interest to forensic neuropathology. The expression of an epitope sometimes only provides information on the current functional state of a cell. Activated astrocytes, for example, are GFAP and/or vimentin positive, activated microglia CD68 positive, whereas in a resting state they are partly GFAP negative and CD68 negative (see above).

Surveys of immunohistochemical methods have been published by Sternberger (1979) and, more re- cently, by DeLellis (1988). Only a few antibodies used for routine demonstration of cytological and histological structures will be described here (Table 5.4).

Details on the numerous other staining methods can be obtained from the relevant volumes dealing with histological techniques (Luna 1960; Böck 1989).

Here the following histochemical methods will be de- scribed:

▬ Demonstration of glycoproteins using periodic acid

▬ Demonstration of iron III (hemosiderin as a cata- bolic product of hemoglobin) using Prussian blue reaction

▬ Fat (lipid) demonstration using oil red O or Su- dan solution

5.3

Postmortem Imaging

Radiographic study of MBI, of gunshot wounds of the head in particular, has long be used for localization and documentation of skull fractures as well as of bone and missile fragments in the brain (Lorenz 1948; Metter et al. 1989; for review see Brodgon 1998;

Brodgon and Lichtenstein 2000). Cerebral computed tomography (CCT) is often employed in forensic investigations of, for example, gunshot wounds of the head (Schumacher et al. 1983a, b, 1985, 1987; Karger et al. 1998) and hanging (Wallace et al. 1994). CT should commonly be applied in forensic traumatology for biometric analysis and reconstruction (Donchin et al. 1994; Farkash et al. 2000; Thali et al.

2001). Such studies, like those of MBI (Schumacher et al. 1987; Wallace et al. 1994), are sometimes performed on isolated, formalin-fixed brains (Chance et al. 1998). Helical CCT scan, by contrast, has been used for 3D imaging of skull fractures (Myers et al. 1999). The brain can also be studied with mag- net resonance imaging (MRI), whose findings correlate closely with neuropathological findings and are highly reproducible in isolated, formalin-fixed brains (sensitivity, 83.39%; specificity, 76.6%) (van den Hauwe et al. 1995). Several authors have shown that postmortem imaging findings correlate with known neurological disease patterns (Nagara et al.

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1987; Wind et al. 1988; Bisset 1998; Erly et al. 1999).

Harris (1999) demonstrated the utility of such imaging studies for forensic pathology (Oehmichen et al.

2003, see also Chapter 8) (Figs. 8.15−8.18).

Continued technical improvements have in- creased the sensitivity of imaging procedures in postmortem examinations (Gean 1995; Gilman 1998;

Gulyas and Lestar 1999). These techniques are in- creasingly in demand as society grows less tolerant toward the conduction of autopsies. Even if autopsy remains the „gold standard“ of pathologic-anatomi- cal diagnosis and forensic investigation, imaging procedures can be the source of valuable supplementary information. In cases where they are able to produce results superior to those of other methods, imaging procedures should be applied on a regular basis. They can be used in the quality control of the results of postmortem examination and its documentation, by for example localizing foreign objects (bone splinters, projectiles, and other types of foreign object). 3D reconstruction using digital imaging enables among other possibilities biomechanical reconstruction of a traumatic event postmortem (cf.

Wind et al. 1988).

When does CCT/MRI imaging provide useful information for the forensic neuropathologist?

1. Providing information on the brain in surviving victims of gunshot injury. Such findings, for ex-

ample, are sometimes needed for court proceed- ings.

2. The pathologist may review the imaging together with the radiologist after a patient has died in surgery that has resulted in excision of the relevant portions (in the case of gunshot wounds:

entrance, exit, dura, bone, and portions of the injured brain) of the deceased‘s head.

3. Review of the imaging with the radiologist can be helpful if a patient has survived an injury for weeks, months or years and the brain at death ex- hibits only repair and scarring.

4. Review of premortem imaging can be helpful if the brain has undergone autolysis in situ, as hap- pens in „respirator brain,“ for example, which results in the bullet descending through the liq- uefying brain to the lowermost intracranial location.

5. Postmortem CCT/MRI may help in the examination of bodies burned so severely that bony entrances have been obscured, damaged or obliter- ated altogether.

6. Imaging of facial fractures so as to obviate the need of disturbing the features by dissection.

7. Examination of embalmed, exhumed bodies in which entrances, exits, and trajectories are altered.

Table 5.6. Selected antibodies for identification of specific cell types, their processes and/or specific types of injury

Target Epitope Author

Neuron Neuron-specific enolase (NSE) (Dako) Reynolds et al. (1994)

Axons

(Normal) Neurofilament protein (NFP) (Binding site) Perez et al. (1990) (Pathologic) β-Amyloid precursor protein (β-APP) (Chemicon) Gentleman et al. (1993) Dendrites Microtubule-associated protein (MAP) (Dianova) Li et al. (1997)

Myelin Myelin basic protein (MBP) (Dako) Hardy et al. (1996)

Synapses Synaptophysin (Binding site) Reynolds et al. (1994)

Astrocyte Glial fibrillary acidic protein (GFAP) (Dako) Galou et al. (1996)

Vimentin (Dako) Reynolds et al. (1994)

S 100 protein (Binding site) Reynolds et al. (1994)

Oligodendrocyte RIP (Sigma) Friedman et al. (1989)

Carbonic anhydrase II (CA II) (binding site) Cammer et al. (1977) Microglia

(Normal) F4/80 (Serotec) Gordon et al. (1992)

(Pathologic) CD68 (Dako) Esiri and McGee (1986)

Endothelial cells Factor VIII (F VIII) (Dako) Sehested and Hou-Jensen

(1981)

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In light of these advantages there can be no ques- tion of the importance of imaging procedures for evaluation in neurotraumatology, above all for the examination and documentation of gunshot wounds of the head. The value of imaging techniques for postmortem documentation of gunshot injuries of the head is evident, especially if bone or projectile fragments have been displaced at autopsy. Biometric reconstruction using the digital data obtained with imaging procedures on the isolated, formalin-fixed brain is able to replicate the position of the brain in a standing individual (see pp. 167 ff).

These observations allow the following conclu- sions:

▬ CCT and MRI are no substitute for autopsy in medicolegal cases. Rather they should be used to supplement other findings in cases where survival or interventions have altered the original wounds.

▬ 3D imaging in most cases is based on spiral CCT volume data sets and is able to establish the direction in which the missile traveled in gunshot injuries, tracing the missile track, and distinguish- ing between entrance and exit wounds. Com- mercially available software is able to process the biometrically relevant data. Lesions along the missile track such as edema and hemorrhage can be distinguished by MR imaging of the brain parenchyma, thus providing important information on primary and secondary changes caused, for example, by gunshot wounds of the head.

▬ CCT is especially helpful in locating opaque foreign objects in the brain which are sometimes difficult to locate at autopsy, objects such as bullet and bone fragments.

▬ Although autopsy on the isolated, formalin-fixed brain can also demonstrate opaque fragments and the missile track, it is usually not able to de- termine the direction of travel, making biometric classification difficult or impossible.

▬ CCT-obtained 3D imaging is positively indicated if there is uncertainty regarding the location of entrance and exit wounds, or where the bullet and/or bullet fragments have lodged in the brain, and if documentation of the in situ locations of bone and projectile fragments is needed. CCT- obtained 3D imaging is relatively indicated if photographic documentation is impossible.

▬ Imaging procedures have the disadvantage com- pared to autopsy of being less able to detect microscopic signs of a preexisting disease process or hypoxic changes to neuronal tissues. They are also unable to assist in assessing the vitality of gunshot wounds or of survival time.

Bibliography

Courville CB (1964) Forensic neuropathology. Lesions of the brain and spinal cord of medicolegal importance. Callaghan, Mun- delein, Ill.

Hill RB, Anderson RE (1988) The autopsy: medical practice and public policy. Butterworths, Boston, Mass.

References

Auer RN, Krcek J, Butt JC (1994) Delayed symptoms and death following minor head trauma with occult vertebral artery injury.

J Neurol Neurosurg Psychiatry 57:500−502

Berzlanovich AM, Sinn E, Muhm MA (1998) Technique for dissecting the cervical vertebral column. J Forensic Sci 43:190−193 Bisset R (1998) Magnetic resonance imaging may be alternative to

necropsy. Br Med J 317:1450

Böck P (1989) Romeis mikroskopische Technik. Urban und Schwar- zenberg, Munich

Bond WW, Favero MS, Petersen NJ et al. (1981) Survival of hepatitis B virus after drying and storage of one week. Lancet 1:550−551

Brodgon BG (1998) Forensic radiology. CRC, Boca Raton, Fla.

Brodgon BG, Lichtenstein JE (2000) Forensic radiology in histori- cal perspective. Crit Rev Diagn Imaging 41:13−42

Brown P, Gibbs GJ, Gadjusek DC et al. (1986) Transmission of Creutzfeldt-Jakob disease from formalin-fixed, paraffin-embedded human brain tissue. N Engl J Med 315:1614−1615 Cajal SR (1913) Sobre un nuevo proceder de impregnación de la

neuroglia y sus resultados en los centros nerviosos del hom- bre y animales. Trab Lab Invest Biol Univ Madrid 11:219−252 Cammer W, Bieler L, Fredman T, Norton WT (1977) Quantitation of

myelin carbonic anhydrase: development and subfraction- ation of rat brain myelin and comparison with myelin from other species. Brain Res 138:17−28

Centers for Disease Control and Prevention (1998) Recommen- dations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. Morb Mort Wkly Rep 47:1−39

Chance SA, Gerig G, Highley JR, McDonald B (1998) Validation of MRI cortical surface rendering on fixed human post-mortem brain. J Neuropathol Appl Neurobiol 24: 149−150

Conn HJ, Darrow MA (1943) Staining procedures. Biotech Publica- tions, Geneva

Courville CB (1964) Forensic neuropathology. Lesions of the brain and spinal cord of medicolegal importance. Callaghan, Mun- delein, Ill.

Dawson TP, Neal JW, Llewellyn L, Thomas C (2003) Neuropathol- ogy techniques. Arnold, London

DeLellis RA (1988) Advances in immunohistochemistry. Raven, New York

Donchin J, Rivkind AI, Bar-Ziv J et al. (1994) Utility of postmortem computed tomography in trauma victims. J Trauma 37:552−556

(11)

Erly WK, Bellon RJ, Seeger JF, Carmody RF (1999) MR imaging of acute coccidioidal meningitis. Am J Neuroradiol 20:509−514 Esiri MM, McGee JO (1986) Monoclonal antibody to macrophages

(EMB/11), labels, macrophages and microglial cells in human brain. J Clin Pathol 39:615−621

Farkash U, Scope A, Lynn M et al (2000) Preliminary experience with postmortem computed tomography in military pen- etrating trauma. J Trauma 48:303−309

Feigin I, Cravioto H (1961) A histochemical study of myelin. A dif- ference in the solubility of the glycolipid components in the central and peripheral nervous systems. J Neuropathol Exp Neurol 20:245−254

Friedman B, Hockfield S, Black JA et al. (1989) In situ demonstration of mature oligodendrocytes and their processes: an im- munocytochemical study with a new monoclonal antibody, rip. Glia 2:380−390

Galou M, Colucci-Guyon E, Ensergueix D et al. (1996) Disrupted glial fibrillary acidic protein network in astrocytes from vi- men knockout mice. J Cell Biol 133:853−863

Gean AD (1995) Imaging of head trauma. Raven, New York Gentleman SM, Nash AJ, Sweeting CJ et al. (1993) β-Amyloid pre-

cursor protein (β-APP) as a marker of axonal injury after head injury. Neurosci Lett 160:139−144

Gerszten PC, Martínez AJ (1995) The neuropathology of South American mummies. Neurosurgery 36:756−761

Gilman S (1998) Imaging the brain. N Engl J Med 338:812−820 (I);

338:889−896 (II)

Gomori G (1937) Silver impregnation of reticulum in paraffin sections. Am J Pathol 13:993−1002

Gordon S et al (1992) Antigen markers of macrophage differentia- tion murine tissues. Curr Top Microbiol Immunol 181:1−37 Gulyas B, Lestar M (1999) Functional neuroimaging-functional

neuropathology: the quest for validation. Neuroreport 10:R1 Hardy RJ, Lazzarini RA, Colman DR, Friedrich VL (1996) Cytoplas- mic and nuclear localization of myelin basic proteins reveals heterogeneity among oligodendrocytes. J Neurosci Res 46:246−257

Harris LS (1999) Postmortem magnetic resonance images of the injured brain: effective evidence in the courtroom. Forensic Sci Int 50:179−185

Hauwe L van den, Parizel PM, Martin JJ et al. (1995) Postmortem MRI of the brain with neuropathological correlation. Neuro- radiology 37:343−349

Hill RB, Anderson RE (1988) The autopsy − medical practice and public policy. Butterworths, Boston, Mass.

Hirano A, Zimmermann HM (1962) Silver impregnation of nerve cells and fibers in celloidin sections. Arch Neurol 6:114−122 Holzer W (1921) Über eine neue Methode der Gliafaserfärbung. Z

Ges Neurol 69:354−363

Johnson RT, Gibbs CJ (1998) Medical process: Creutzfeldt-Jakob disease and related transmissible spongiform encephalopathies. N Engl J Med 339:1994−2004

Karger B, Puskas Z, Ruwald B et al. (1998) Morphological findings in the brain after experimental gunshots using radiology, pathology and histology. Int J Legal Med 111:314−319 Kimura A, Ikeda H, Yasuda S et al. (1995) Brain tissue identifica-

tion based on myosin heavy chain isoforms. Int J Legal Med 107:193−196

Klüver H, Barrera E (1953) A method for the combined staining of cells and fibers in the nervous system. J Neuropathol Exp Neurol 12:400−403

Li GL, Ahlgren S, Farooque M et al. (1997) Lesions of axons and dendrites in spinal cord trauma. In: Oehmichen M, König HG (eds) Neurotraumatology − biomechanic aspects, cyto- logic and molecular mechanisms. Schmidt-Römhild, Lübeck, pp 187−201

Lorenz R (1948) Der Schusskanal im Röntgenbilde. Dtsch Z Geri- chtl Med 39:435−448

Luna LG (ed) (1960) Manual of histologic staining methods of the Armed Forces Institute of Pathology. McGraw-Hill, New York Matsui T, Hirano A (1978) An atlas of the human brain for comput-

erized tomography. Igaku-Shoin, Tokyo, pp 1−570

Metter D, Stute H, Rauschke J (1989) Rechtsmedizinische Röntge- nuntersuchungen bei Schädelschüssen. Beitr Gerichtl Med 47:473−477

Miller DC (1988) Creutzfeldt-Jakob disease in histopathology technicians. N Engl J Med 318:853−854

Myers JC, Okoye MI, Kiple D et al. (1999) Three-dimensional (3-D) imaging in post-mortem examination: elucidation and identification of cranial and facial fractures in victims of homicide utilizing 3-D computerized imaging reconstruction techniques. Int J Legal Med 113:33−37

Nagara H, Inoue T, Koga T et al. (1987) Formalin fixed brains are useful for magnetic resonance imaging (MRI) study. J Neurol Sci 81:67−77

Naoumenko J, Feigin I (1963) A modification for paraffin sections of silver carbonate impregnation for microglia. Acta Neuro- pathol (Berl) 2:402−406

Nolte KB, Taylor DG, Richmond JY (2002) Biosafety considerations for autopsy. Am J Forensic Med Pathol 23:107−122

Oehmichen M (1984) Fortschritte zytologischer Methoden in der forensischen Pathologie. Pathologe 5:200−203

Oehmichen M, König HG, Pedal I (1984) Zytologischer Befund als Indiz: morphologische und immunhistochemische Iden- tifzierung von menschlichem Hirngewebe an der Täterkleid- ung. Arch Kriminol 173:129−141

Oehmichen M, Gehl H-B, Meissner C et al (2003) Forensic pathological aspects of postmortem imaging of gunshot injury to the head. Documentation and biometric data. Acta Neuro- pathol (Berl) 105:570−580

Perez MA, Saul SH, Trojanowski JQ (1990) Neurofilament and chromogranin expression in normal and neoplastic neuro- endocrine cells of the human gastrointestinal tract and pan- creas. Cancer 65:1219−1725

Rális HM, Beesley RA, Rális ZA (1973) Techniques in neurohistol- ogy. Butterworths, London

Reynolds GM, Young FI, Young JA et al. (1994) Microwave oven antigen retrieval applied to the immunostaining of cytopathology specimens. Cytopathology 5:345−358

Schulze-Schaeffer WJ, Giese A, Kretzschmar HA (1998) Creutzfeldt- Jakob-Krankheit − neue Aspekte für die Rechtsmedizin. Re- chtsmedizin 8:123−129

Schumacher M, Oehmichen M, König HG (1983a) The computed tomography as a helpful method in forensic medicine for the reconstruction of gunshot lesions of the brain. In: Villani R et

(12)

al (eds) Advances in neuro-traumatology. Excerpta Medica, Amsterdam, pp 198−200

Schumacher M, Oehmichen M, König HG, Einighammer H (1983b) Intravitale und postmortale CT-Untersuchungen bei Hirn- schussverletzungen. Fortschr Röntgenstr 139:58−62 Schumacher M, Oehmichen M, König HG et al. (1985) Computer-

tomographische Untersuchungen zur Wundballistik krani- aler Schussverletzungen. Beitr Gerichtl Med 43:95−101 Schumacher M, Oehmichen M, Hassler W (1987) Intravitale CT-Be-

funde bei kranialen Schussverletzungen. In: Kohlmeyer K (ed) Aktuelle Probleme der Neurotraumatologie und Klinischen Neuropsychologie. Biermann, Regensburg

Sehested M, Hou-Jensen K (1991) Factor VIII-related antigen as an endothelial cell marker in benign and malignant diseases.

Virchows Arch Pathol Anat 391:217−225

Shapiro CN (1995) Occupational risk of infection with hepatitis B and hepatitis C virus. Surg Clin North Am 75:1047−1056 Sternberger LA (1979) Immunocytochemistry, 2nd edn. Wiley,

New York

Thali MJ, Watzke O, Kachelriess M et al. (2001) Bullets and metal artifact − “state of the art.” Rechtsmedizin 11:193

Vonsattel JPG, Aizawa H, Ge P, DiFiglia M et al (1995) An improved approach to prepare human brains for research. J Neuro- pathol Exp Neurol 54:42−56

Wallace SK, Cohen WA, Stern EJ, Reay DT (1994) Judicial hanging:

postmortem radiographic, CT, and MR imaging features with autopsy confirmation. Radiology 193:263−267

Wind G, Ginley RW, Rich NM (1988) Three-dimensional computer graphic modeling of ballistic injuries. J Trauma 28:S16−S20

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Physical Trauma II

Chapter 6

Basic Principles of Mechanical Trauma 97 Chapter 7

Injuries of the Brain’s Coverings 111 Chapter 8

Open Brain Injuries 152 Chapter 9

Closed Brain Injuries 177 Chapter 10

Injuries of Spine and Spinal Cord 217 Chapter 11

Injuries of Peripheral Nerves 241 Chapter 12

Special Physical Trauma 245