PMC7683776_01
PMC7683776_02
Interictal electroencephalogram of the 64-days-old puppy with neonatal encephalopathy with seizures. The average potential reference method was used. Polyspikes (A, arrows) and isolated spikes (B, arrows & arrowheads) were observed. In addition, alpha-band rhythms consisted of spikes and sharp waves (C, surrounded by the rectangle) were also seen.
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F1
Lactate and lipid peaks were detected (Supplementary Figure 1), which are not typically seen in normal brain tissue, although lactate is known to be present in the cerebrospinal fluid (CSF).
PMC7683776_03
Interictal electroencephalogram of the 64-days-old puppy with neonatal encephalopathy with seizures. The average potential reference method was used. Polyspikes (A, arrows) and isolated spikes (B, arrows & arrowheads) were observed. In addition, alpha-band rhythms consisted of spikes and sharp waves (C, surrounded by the rectangle) were also seen.
fvets-07-578936-g0001.jpg
PMC7683776_03_fvets-07-578936-g0001.jpg
F1
Lactate and lipid peaks were detected (Supplementary Figure 1), which are not typically seen in normal brain tissue, although lactate is known to be present in the cerebrospinal fluid (CSF).
Magnetic resonance T2-weighted imaging of the brain in the median sagittal plane of the patient (64-days-old) (A) and a normal beagle dog (73-days-old) as a similar-age reference (B). (A) Dilation of the third and fourth ventricles, presumptive dilatation of the pineal recess or a quadrigeminal cyst, and diffuse hypoplasia of corpus callosum, rostral commissure, caudal commissure, and the ventral part of the cerebellar vermis (lingula, nodulus, and uvula of the cerebellum) are seen. The reduced size of the brainstem and cerebellum and thinning of the interthalamic adhesion are also seen. (B) Unlike the patient, corpus callosum (black arrow), rostral commissure (yellow arrow), and caudal commissure (yellow arrowhead) are clearly recognized in a normal beagle dog (73-days-old). (C) Diffusion tensor imaging (DTI) tractography of the patient's brain. Regions of interest were placed to visualize corpus callosum. There seemed to be no visible commissural fiber across both hemispheres. This finding demonstrated hypoplasia of corpus callosum, although there is a possibility that DTI tractography may not accurately represent the white matter structure.
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Supplementary Appendix 1. Since the patient was a 64-days-old puppy and the brain should be immature, conventional MRI previously obtained from a 73-days-old normal Beagle dog as a part of another study was used as a reference (Figures 2B, 3D-F). The MRI showed the reduced size of the whole brain including the cerebellum and brainstem, dilation of the ventricular system, presumptive dilatation of the pineal recess or an arachnoid diverticulum, subcortical white matter hypoplasia, and diffuse hypoplasia of corpus callosum, rostral commissure, caudal commissure, and the ventral part of the cerebellar vermis (lingula, nodulus, and uvula of the cerebellum) (Figures 2A, 3A,B). Those findings were not recognized in MRI of a 73-days-old normal Beagle dog used as a reference (Figures 2B, 3D,E). Functool which is an internal software of the MRI unit (GE Healthcare) supported the diffuse hypoplasia of the corpus callosum (Figure 2C). Fractional anisotropy (FA) values of the anterior internal capsule and the posterior internal capsule, which were calculated by DTI, were ~0.43 and 0.415, respectively.
Magnetic resonance T2-weighted imaging (T2WI) of the brain in the transverse plane of the patient (64-days-old) (A-C) and a normal beagle dog (73-days-old) as a similar-age reference (D-F). On the T2WI at the levels of the frontal lobe of the patient (A,B), the lateral ventricles seemed to have upturned pointed corners (yellow arrows). Marked hypoplasia of subcortical white matter is also seen (yellow arrowheads). The brain sulci are clearly visible due to the suspected developmental abnormality. On the T2WI at the level of the occipital lobe of the patient (C), there are T2-hyperintense lesions (blue arrows) in the right and left white matter in addition to the dilated left lateral ventricle. Corpus callosum (E, black arrow) is clearly seen in a 73-days-old normal beagle dog (E), but not in the patient (B).
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Supplementary Appendix 1. Since the patient was a 64-days-old puppy and the brain should be immature, conventional MRI previously obtained from a 73-days-old normal Beagle dog as a part of another study was used as a reference (Figures 2B, 3D-F). The MRI showed the reduced size of the whole brain including the cerebellum and brainstem, dilation of the ventricular system, presumptive dilatation of the pineal recess or an arachnoid diverticulum, subcortical white matter hypoplasia, and diffuse hypoplasia of corpus callosum, rostral commissure, caudal commissure, and the ventral part of the cerebellar vermis (lingula, nodulus, and uvula of the cerebellum) (Figures 2A, 3A,B). Those findings were not recognized in MRI of a 73-days-old normal Beagle dog used as a reference (Figures 2B, 3D,E). T2-weighted imaging (T2WI) of the lateral ventricles seemed to show upturned pointed corners (Figures 3A,B). This finding is often seen in canine patients with corpus callosum aplasia. T2WI and fluid-attenuated inversion recovery (FLAIR) hyperintensity and T1-weighted imaging (T1WI) hypointensity, not enhanced with gadodiamide, was observed bilaterally in the occipital white matter and more evident in the left hemisphere (Figure 3C). This region showed an increased apparent diffusion coefficient (ADC) value (1.8 x 10-3 mm2/s), which was calculated by diffusion-weighted imaging, and a decreased FA value (0.15) when compared with the previously reported values from normal 9-week-old dogs [ADC, approximately 0.9 x 10-3 mm2/s (the subcortical white matter); FA, approximately 0.475 and 0.34 (the anterior internal capsule and the posterior internal capsule, respectively)]. ADC value, and decreased FA value in MRI (Figure 3C). Although less apparent than in the occipital lobe, rarefaction with hypomyelination was also identified in the frontal lobe histopathologically.
Gross and histopathological necropsy findings. (A) A diagonal view of the fixed brain shows hypoplasia of the cerebellum. The cerebellar vermis is markedly small. (B) An HE-stained section of the cerebellum shows clusters of ectopic cells in the cerebellar cortex. Bar = 500 mum. (C) An HE-stained section of the cerebrum shows clusters of cells in the wall of lateral ventricle in the frontal lobe (arrows). Bar = 500 mum. (D) Luxol fast blue and HE-stained section of the cerebrum shows rarefaction of the white matter with hypomyelination (asterisks). Bar = 500 mum.
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Gross examination showed hypoplasia of the ventral part of the cerebellar vermis (Figure 4A) and dilation of the left lateral ventricle. These findings seen in the cerebellar cortex were consistent with the previously reported findings in NEwS (Figure 4B). Undifferentiated cells were multifocally or diffusely distributed in the subventricular zone of the frontal lobe (Figure 4C).