Focal white matter changes in spasmodic dysphonia: a combined diffusion tensor imaging and neuropathological study

Abstract

Spasmodic dysphonia is a neurological disorder characterized by involuntary spasms in the laryngeal muscles during speech production. Although the clinical symptoms are well characterized, the pathophysiology of this voice disorder is unknown. We describe here, for the first time to our knowledge, disorder-specific brain abnormalities in these patients as determined by a combined approach of diffusion tensor imaging (DTI) and postmortem histopathology. We used DTI to identify brain changes and to target those brain regions for neuropathological examination. DTI showed right-sided decrease of fractional anisotropy in the genu of the internal capsule and bilateral increase of overall water diffusivity in the white matter along the corticobulbar/corticospinal tract in 20 spasmodic dysphonia patients compared to 20 healthy subjects. In addition, water diffusivity was bilaterally increased in the lentiform nucleus, ventral thalamus and cerebellar white and grey matter in the patients. These brain changes were substantiated with focal histopathological abnormalities presented as a loss of axonal density and myelin content in the right genu of the internal capsule and clusters of mineral depositions, containing calcium, phosphorus and iron, in the parenchyma and vessel walls of the posterior limb of the internal capsule, putamen, globus pallidus and cerebellum in the postmortem brain tissue from one patient compared to three controls. The specificity of these brain abnormalities is confirmed by their localization, limited only to the corticobulbar/corticospinal tract and its main input/output structures. We also found positive correlation between the diffusivity changes and clinical symptoms of spasmodic dysphonia (r = 0.509, P = 0.037). These brain abnormalities may alter the central control of voluntary voice production and, therefore, may underlie the pathophysiology of this disorder.

Figures

Figure 1

Group differences in fractional anisotropy. (A) TBSS whole-brain and (B) a priori ROI analyses found significant FA decrease in the right genu of the internal capsule in SD patients. Group differences (Patients < Controls) are overlaid onto the average FA map across all subjects; plane coordinates of axial brain images are in Talairach-Tournoux standard space, respectively; color bar indicates the significance range at Z > 3.2. Box plots indicate median and upper and lower quartiles. Error bars indicate the range between the 90th and 10th percentiles. Asterisk indicates significant difference between two groups. R - right; L - left.

Figure 2

Group differences in Trace (D). (A) TBSS whole-brain analysis found significant increase of overall diffusivity in the corona radiata, genu and posterior limb of the internal capsule, cerebral peduncle, ventral thalamus, and cerebellum. Group differences (Patients > Control) are overlaid onto the average FA map across all subjects; plane coordinates of axial brain images are in Talairach-Tournoux standard space, respectively; color bar indicates the significance range at Z > 3.2. (B) A priori ROI analysis found significant increase of Trace values (× 10-3 mm2/sec) in the genu (GIC) and posterior limb (PLIC) of the internal capsule, lentiform nucleus (LN) and ventral thalamus (VTh) with a trend in the middle cerebellar peduncle (MCP) in SD patients. Box plots indicate median and upper and lower quartiles. Error bars indicate the range between the 90th and 10th percentiles. Asterisks indicate significant difference between two groups. R - right; L - left.

Figure 3

Correlation between diffusion parameters. (A) Significant inverse correlation between overall mean values of fractional anisotropy (FA) and Trace (D) was stronger in SD patients than in healthy volunteers (HV). (B) Significant inverse correlation between the FA and Trace values were found in the genu of the internal capsule. (C) Positive significant correlation was determined between the Trace values in the ventral thalamus (VTh) and number of breaks in SD patients.

Figure 4

DTI-guided postmortem neuropathological examination of FA changes in the genu of the internal capsule. (A) An example of T1-weighted image in coronal plane with overlaid FA group differences (left) and visually matched postmortem coronal brain slice (right). The black box indicates the region of tissue extraction from the postmortem specimen. (B) Photomicrograph of the control sample shows well-organized white matter in the genu of the internal capsule (GIC) and (C) reduced white matter density (*) in the GIC in an SD patient (H&E stain). Area of reduced white matter density in the GIC reveals (D) reduction of myelin content (LFB/PAS stain), (E) diffusely scattered reactive microglial/macrophage cells (KP1 stain), and (F) moderate reduction of axonal density (anti-NFTP stain).

Figure 5

DTI-guided postmortem neuropathological examination of Trace (D) changes in an SD patient. Photomicrographs of the mineral depositions (dark-blue/black visualization product) in the parenchyma of the putamen (A) and globus pallidus (B) and in the vessel wall in the posterior limb of the internal capsule (C) (H&E stain). An example from putaminal tissue shows accumulations of phosphorus (von Kossa stain) (D) and calcium (alizarin red S stain) (E) in the parenchyma and deposition of iron in the vessel wall (Prussian blue stain) (F) with scattered single iron-positive cells in the parenchyma (arrowheads).

Figure 6

Scanning electron microscopy with energy dispersive X-ray analysis (SEM-EDXA) of tissue depositions in an SD patient. (A) Photomicrograph of the deposition (*) in the putaminal parenchyma on H&E stain (I) and SEM (II) (scale 100 μm). EDXA shows accumulation of phosphorus (P) and calcium (Ca). (B) Vessel wall deposition in the putamen (*) shown on H&E stain (I) and SEM (II) (scale 50 μm). EDXA detected collocation of phosphorus (P), calcium (Ca), and iron (Fe).

Figure 7

Simplified schematic illustration of the neural network of voluntary laryngeal control in humans. Direct projections from the laryngeal motor cortex (LM1) to the phonatory motor nuclei (nucleus ambiguus, NA) descend via the corticobulbar/corticospinal tract (CBT/CST). Several connections exist between the LM1 and the subcortical motor system. The putamen (Put) receives input from the LM1 and projects back to the LM1 via the globus pallidus (Gp) and ventral lateral thalamus (VTh) forming striato-pallido-thalamo-cortical loop. Cerebellar motor input (Cbl) to the LM1 is via the VTh. Microstructural changes along the CBT/CST as well as in the regions directly or indirectly contributing to the CBT/CST found in this study (dashed areas) may affect voluntary laryngeal control in patients with SD.