LFB/PAS staining of Twitcher mice lacking KC or CXCR2.

<p>Histology of the brains showing LFB staining (blue) and PAS staining (pink) in the corpus callosum (A-D) and cerebellum (E-H) in the wildtype, Twitcher, KC−/−GALC−/− mice and CXCR2−/−GALC−/− mice. The KC−/−GALC−/− forebrain (C) and cerebellum (G) show histology which is essentially identical to the Twitcher mice (KC+/+GALC−/−; B and F) with similar myelin staining and distribution of globoid cells. The CXCR2−/−GALC−/− mouse brains also demonstrate similar histology (D, H) compared to the Twitcher mice (B, F). There is no apparent difference between the KC+/+GALC+/+ and KC−/−GALC+/+ or the CXCR2−/−GALC+/+ mice in all the sections examined (images not shown). Scale bar equals 50 µm.</p

Flow cytometric characterization of inflammatory cells in the KC−/−GALC−/− brains.

<p>The upper row of Panel A contains representative bivariate contour plots showing CD8 and CD4 T-cells at day 36 in various groups of mice. The lower row in panel A contain representative bivariate contour plots showing activated microglia (CD45^hi CD11b+, upper right quadrant) isolated from the brain at 36 days of age. There is no significant increase in CD8+ T-cells in KC−/−GALC+/+, KC+/+GALC−/− or KC−/−GALC−/− mice compared to the KC+/+GALC+/+ mice (B). There is no significant difference in the CD4+ T-cells in the brains of the KC−/−GALC−/− and KC+/+GALC−/− mice compared to the KC+/+GALC+/+ mice (C). There is no significant difference between the KC+/+GALC−/− mice and KC−/−GALC−/− mice in the number of activated microglia (D).</p

Altered cytokine and growth factor levels in the spinal cord of Twitcher mice that could possibly compensate for the lack of KC or CXCR2.

<p>MIP-2 (CXCL2) (A), PDGF-BB (B), and FGF-2 (C) levels are significantly and progressively elevated in the spinal cords of the Twitcher mice. Vertical bars represent the mean and the error bars represent one SEM (**p<0.01, ***p<0.001).</p

Cytokine and chemokine levels in the CNS of Twitcher mice.

<p>The fold-elevation of various cytokines/chemokines in the brain is shown. Among all the assayed molecules, the chemokine KC showed the greatest fold change in the brains (>15-fold increase) of the Twitcher mice (A). The levels of KC in the brains (B) and the spinal cords (C) of the Twitcher mice showed a progressive increase with time. The vertical bars represent the means and the error bars represent one SEM; **p<0.01, ***p<0.001.</p

Age-related progressive loss of Purkinje cells in the cerebellar lobules in MPS IIIB mice.

<p>ANOVA of Purkinje cell count by lobe showed that old MPS IIIB mice (white bars) had significantly lower neuronal counts across cerebellar lobes compared to old heterozygous controls (black bars) although differences between groups were lobe dependent. Pairwise comparisons conducted in the older cohort of mice showed that differences between groups were greatest in lobes VI (*p = 0.001), IV/V (*p = 0.003), VIII (*p = 0.004), IX (*p = 0.011), and III (*p = 0.036). An ANOVA of the Purkinje cell count data that included both young and old cohorts of mice resulted in a significant main effect of Age (p<0.001) and a significant Age by Genotype interaction (p = 0.003). Other important effects included significant Lobe by Genotype (p = 0.010), and Lobe by Age (p<0.001) interactions. Additional comparisons showed that the older MPS IIIB mice had significantly fewer neurons across the cerebellar lobes compared to the younger MPS IIIB mice (p<0.001, (p<0.006, the Bonferroni corrected value)), while no differences were found between older and younger control mice.</p

Older MPS IIIB mice are impaired on the rocking rotarod.

<p>Mean duration (in seconds) of the time spent on the rocking rotarod as a function of increasing ages (days) for MPS IIIB (open squares) and heterozygous (filled diamonds) control mice. No performance differences were observed between groups on the rocking rotarod in the younger cohort of mice (left panel). In contrast, an ANOVA conducted on the first 4 data points from the older cohort of mice (right panel) revealed a significant main effect of Genotype (p = 0.030), thus showing, in general, that the older MPS IIIB mice, remained on the rod for a significantly shorter time compared to the heterozygous control mice. Differences between groups were greatest when the mice were tested at 244 (*p = 0.013) and 302 (†p = 0.034) days of age. Even greater deficits were observed in the MPS IIIB mice when they were tested at older ages although small sample sizes resulting from high mortality rates in the MPS IIIB mice precluded formal statistical analyses. Numbers in parentheses represent sample sizes at specific ages of testing. The dotted line indicates that the last two test sessions (327 and 372 days) were not included in the overall ANOVA since sample sizes were so small due to increased mortality.</p

Inner ear pathology in MPS IIIB mice at 30 wks.

<p>A. Radial view of cochlear upper basal turn showing grossly normal features. B. Crista ampullaris of lateral semicircular canal in the same animal shows abnormal lysosomal storage in both hair cells and supporting cells of the sensory epithelium (black arrow), and in dark cells (white arrow). C. Expanded view of medial organ of Corti in A. The only cells showing storage are inner pillar cells (IP) (black arrow). Mesothelial cells lining the basilar membrane (BM) show the aberrant storage (white arrowheads), as do adherent inflammatory cells (black arrowheads). Vacuoles within inner hair cells (asterisk, IHC) are probably a processing artifact. D. Spiral ganglion cell region from A shows aberrant storage only in glial cells (white arrowheads). E. Spiral ligament and lateral organ of Corti from A shows aberrant storage in outer sulcus cells (white arrowhead, OSC), epithelial cells of spiral prominence (black arrowhead, SP), Type III fibrocytes (white arrows, T3), and Type II fibrocytes (black arrows, T2). Type IV fibrocytes (T4) also showed storage, not apparent in this view. F. Complete loss of hair cells and other differentiated cell types of the organ of Corti (black arrow), with secondary loss of neuronal processes (white arrow). This was more prevalent in MPS IIIB mice than in WTs. G. Plot of the number of hair cell profiles versus neuronal density, as seen in radial view in the lower base for 5 MPS IIIB and 5 WT mice at 30 wks. Differences in hair cell numbers by genotype are highly significant (t-test, p<.001). Because inner hair cells were less affected than outer hair cells, only numbers <1.0 indicate loss of IHCs. Neuronal density decreases only when IHCs are missing, so that neuronal loss appears secondary to hair cell loss in MPS IIIB. StV: Stria vascularis; RM: Reissner's membrane; TM: Tectorial membrane; OHC: Outer hair cells; DC: Deiters' cells; OP: Outer pillar cell; Lim: Spiral limbus.</p

Eye Pathology.

<p>Light micrographs showing pathology from normal (A, F, H) and MPS IIIB (B–D, E, G, I) mouse retinas (A–E), sclera (F, G), and ciliary bodies (H, I). (A) Normal histology from a 4-week old wild type mouse retina is shown and is indistinguishable from older wild type retinas at 45 weeks. (B) In the 8-week old MPS IIIB mouse retina, aberrant lysosomal storage can be seen in non-neuronal cells in the inner retina (arrow). Inset: Localized disruption of the retinal pigment epithelium (RPE) from a nearby region of the same retinal section is shown. (C) At about 16 weeks, the outer segments (OS) of the MPS IIIB mouse are shortened, the outer nuclear layer (ONL) is reduced by 2–4 rows of nuclei, and pyknotic nuclei are seen in the ONL (arrow). Macrophage-like cells are present in the subretinal space (arrowhead). (D) In the 30-week old MPS IIIB mouse retina, OSs are further shortened and the ONL is reduced by nearly half. Inset: higher magnification of the boxed area showing dense, round melanosome-like structures in the RPE. (E) By 34 weeks, a subpopulation of cells in the ganglion cell layer (GCL) has a dense appearance with numerous lysosomal inclusions in the cytoplasm. (F) Normal histology from a 4-week old wild type mouse sclera is shown. (G) Lysosomal storage appears as pale vesicles in the sclera of the MPS IIIB mouse (arrowheads). (H) Normal histology from a 4-week old wild type mouse ciliary body is shown. (I) By 30 weeks of age, the ciliary body of the MPS IIIB mouse appears disorganized and swollen with lysosomal storage vesicles (arrow). Numerous dense, round melanosome-like structures are seen (arrowhead). IS, photoreceptor inner segments; OPL, outer plexiform layer; INL inner nuclear layer; IPL, inner plexiform layer. Smaller bar = 10 micron for A–D, H, I. Larger bar = 10 micron for E–G.</p

Auditory-evoked Brainstem Responses (ABR).

<p>Mean (±SEM) ABR thresholds in MPS IIIB and WT mice at 5, 10, 20 and 40 kHz from age 4 to 45 weeks. Earliest differences by genotype appear at high frequencies (40 kHz). By 30 wks, high frequency hearing losses due to background deficits in C57BL/6 dominate. Threshold differences at lower test frequencies, apparent after 16 wks, probably reflect mixed cochlear and middle ear pathology. Differences by genotype were significant at all ages (p<.001, 2-way ANOVA).</p

Electroretinography.

<p>Mixed (cones and rods) photoreceptor b-wave amplitudes (microvolts) to a light stimulus in dark-adapted wild type (diamonds on dashed line) and MPS IIIB (squares on solid line) mice at ages 4 to 40 weeks demonstrating reduced response in MPS IIIB mice, that is statistically significant from 16 weeks onward. Cone response b-wave amplitudes after light adaptation is depicted for wild type (open diamond/dashed line) and MPS IIIB (open square/solid line) at the same ages and shows no difference in cone response.</p