Neuropathological Findings

The unfixed brain weighed 1350 g. On macroscopic inspection of the cerebral hemi spheres, gyri, sulci and leptomeninges and leptomeningeal blood vessels were normal as were the cranial nerve roots, the brainstem and the cerebellum. An aneurysm of 3 mm diameter was found arising from the anterior communicating artery on the right into the interhemispheric fissure. No herniations were present. Coronal sections showed normal cortex and white matter of the cerebral hemispheres. Except for the dilatation of the lateral ventricles and the findings in the caudate nucleus and putamen (see below), the gray matter of other basal ganglia structures, thalamus, and hypothalamus was unremarkable. Sections of the cerebellum and brainstem showed normal cerebellar folia and white matter, normal dentate nucleus and well-pigmented substantia nigra and locus coeruleus. The spinal cord was not examined.

The striatum showed marked diminution in the size of the caudate nuclei bilaterally, with the nuclei appearing as thin bands of tissue adjacent to the lateral ventricles. The putamen also appeared smaller than normal bilaterally (Fig. 2) .

For histological examination, representative brain regions were fixed in 4% formalin, embedded in paraffin and processed for routine histology. Sections were stained with hematoxylin and eosin, Prussian blue, Luxol-fast blue, Luxol fast blue-PAS, phosphotungstic acid hematoxylin (PTAH), Mallory iron, and Gallyas and Bielschowsky silver impregnation methods. The following primary antibodies

Fig. 2 Macroscopic appearance of a frontal section of the brain of patient WA with McLeod syndrome at the level of the anterior commissure demonstrates the unruptured aneurysm of the anterior communicating artery ("A") and the atrophy of the striatum. In both hemispheres, the small size of the putamen is obvious (small arrows) as well as the shrinkage of the caudate nucleus that is reduced to bands of tissue lining the lateral ventricles (large arrows)

Fig. 2 Macroscopic appearance of a frontal section of the brain of patient WA with McLeod syndrome at the level of the anterior commissure demonstrates the unruptured aneurysm of the anterior communicating artery ("A") and the atrophy of the striatum. In both hemispheres, the small size of the putamen is obvious (small arrows) as well as the shrinkage of the caudate nucleus that is reduced to bands of tissue lining the lateral ventricles (large arrows)

were used for immunohistochemistry: mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (mAb) (DAKO, Glostrup, Denmark), rat anti-a-synuclein mAb (clone 15G7, Connex, Munich, Germany), rabbit anti-ubiquitin antiserum (DAKO) and mouse anti-tau mAb (clone AT8, Innogenetics, Ghent, Belgium). For immunohistochemistry 4 |im sections were deparaffinized in xylene and rehydrated using graded alcohols. To enhance immunoreactivity for a-synuclein sections were boiled in 0.01 M citrate buffer (pH 6.0) in a microwave oven. Sections were incubated in PBS with 2% BSA and 0.01% Triton X-100 at room temperature (RT) for 30 minutes. Incubation with primary antibody was performed at RT for 1 hour. Detection of antibody binding was performed with the alkaline phosphatase anti-alkaline phosphatase system (DAKO) according to the manufacturer's instructions using neufuchsin as chromogen.

Histological examination revealed extensive and marked atrophy of the head of the caudate nucleus, which was shrunken and pale (Fig. 3a), and to a lesser degree of the body of the caudate and the putamen. These affected regions showed severe spongiosis, neuronal loss and astrocytic gliosis (Figs. 3b, c). Within bundles of white matter in the caudate nucleus there was focal loss of axons (Bielschowsky) and of myelin (Luxol fast blue-PAS). PTAH disclosed mild proliferation of glial fibers. The head of the caudate nucleus had a few blue, iron-positive deposits (Mallory). These appeared as a fine line along the surface of erythrocytes within the lumen of blood vessels or on the surface of neural tissue components. The significance of this small amount of iron is not known, but the finding may be an artifact. No macrophages, no cytoplasmic or nuclear inclusions in nerve cells, no abnormal PAS-positive material were observed. Gallyas and Bielschowsky silver stains revealed no abnormal argyrophilic inclusions. In addition, no abnormal labelling was obtained in immunostains for ubiquitin, phosphorylated tau and a-synuclein. Changes in the putamen resembled those in caudate nucleus, but they were less severe and patchy in distribution. Bundles of white matter in the putamen were more severely depleted of myelinated fibers than those in caudate nucleus and

Fig. 3 Histological findings in patient WA with McLeod syndrome (a) Severe atrophy of the head of the caudate nucleus (frontal section; c: caudate nucleus; i.e.: internal capsule;p: putamen), (b) Higher magnification of the caudate with marked astrocytic gliosis and neuronal loss, (c) Anti-GFAP immunostaining showing numerous reactive astrocytes. Scale bars: a, 5 mm, b, and c, 100 | m

Fig. 3 Histological findings in patient WA with McLeod syndrome (a) Severe atrophy of the head of the caudate nucleus (frontal section; c: caudate nucleus; i.e.: internal capsule;p: putamen), (b) Higher magnification of the caudate with marked astrocytic gliosis and neuronal loss, (c) Anti-GFAP immunostaining showing numerous reactive astrocytes. Scale bars: a, 5 mm, b, and c, 100 | m showed a moderately severe loss of axons. The globus pallidus was hypercellular and had a mild, diffuse proliferation of glia, but neurons were not clearly reduced in number. There was a small focus of hypercellularity at the lateral aspect of the right substantia nigra (near the subthalamic nucleus) with small, round nuclei and no visible cytoplasm of the proliferated cells that presumably were astrocytes. A similar but smaller focus of hypercellularity appeared present in the middle of the right thalamus and on the medial aspect of the left thalamus. In the medulla two pink bodies were seen in a nucleus of the tegmentum just superior to the inferior olivary nucleus on one side, possibly corresponding to degenerating neurons.

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