Therapeutic Approaches For Cd

So far therapy for CD is symptomatic. Study on therapy for CD is limited. The development of CD model mouse gives the opportunity to evaluate therapeutic approaches for CD (60,61).

6.1. ASPA Gene Transfer to CD Mouse

Recombinant adenoassociated virus (rAAV) is an interesting gene transfer vehicle, because it is nonpathogenic wild type human parvovirus, all viral genes are removed and replaced by the transgene in the replication-incompetent recombinant version, and it has been shown to be almost completely non toxic after CNS delivery in mammals. Therefore, efficacy of AAV-ASPA injection to the CD mouse was examined (62).

Figure 4. Map of human ASPA containing adeno-associated virus vector.

Recombinant adenoassociated viral construct was prepared at the UFL Gene Vector Core (UFLGTCVC, Gainesville, FL) (Fig.4 ). Plasmid CBA is a CMV-based promoter with a chicken P-actin intervening sequence. The vector was gel purified and a Notl-digested human aspartoacylase or GFP PCR product was cloned. A double cotransfection procedure was used to introduce a rAAV vector plasmid together with pDG-AAV helper plasmid carrying the AAV rep and cap genes, as well as Ad helper genes required for rAAV replication and packaging at a 1:1 molar ratio. The final preparation was having 4.2 x 1011 infectious units/ml (62).

Two ^ of rAAV2-ASPA was stereotaxically injected into the striatum or thalamus of the knockout mouse brain. The gene transfer in the CD mouse brain improved ASPA activity, normalized the NAA level (Fig. 5) and improved spongy degeneration without any side effect even five months after treatment, the maximum period studied. The mean value of ASPA activity in three rAAV-ASPA-GFP injected brains showed 0.116 mU/mg protein, while the uninjected brains had no ASPA activity.

Figure 5. MRI/MRS study of rAAV-hASPA-GFP injected mouse brain. Treatment with AAV-hASPA-GFP normalizes the hyperintensity in the subcortical region, which includes thalamus and subthalamus, in contrast to high signal intensity in the CD mouse. The level of NAA was lower in rAAV-hASPA-GFP treated mouse compared to the CD mouse brain. The reading 2.018 is the location of NAA.

Figure 5. MRI/MRS study of rAAV-hASPA-GFP injected mouse brain. Treatment with AAV-hASPA-GFP normalizes the hyperintensity in the subcortical region, which includes thalamus and subthalamus, in contrast to high signal intensity in the CD mouse. The level of NAA was lower in rAAV-hASPA-GFP treated mouse compared to the CD mouse brain. The reading 2.018 is the location of NAA.

Histopathological study of rAAV-ASPA-GFP injected brain revealed a remarkable decrease in vacuolation in the thalamus AAV, which is probably the structure most affected by the vacuolar spongiform neuropathology in the CD mouse brain (Fig 6A-D). The reduction in vacuolation was seen in 3 as well as 6 months aged mice after rAAV injection. Neuropathology distal to the vector injection sites, such as vacuolation in the hippocampus ot the enlarged ventricles was not affected by the gene transfer (Fig 6 E-H).

6.2. Stem Cell Therapy in the Knockout Mouse

Stem cell therapy is another promising approach for the treatment of neurodegenerative diseases (63). The source of stem cells may be from various sources including bone marrow, amniotic epithelial cells, embryonic layers and brain tissue (6466). Of particular interest are neural stem cells (NSCs) or neural progentitor cells (NPCs) that can be readily isolated from the central nervous system of fetal or postnatal animals (67,68). NSCs are self renewing and multipotent whose differentiative capacity is limited to cells of the central nervous system - neurons, astrocytes and oligodendrocytes (69).

Figure 6. Neuropathology of mouse brain using H&E stain. A. Thalamic vacuolization in an rAAV-GFP-injected 3 months old CD mouse survived until 6 months of age, B. survived until 5 months of age, C. survived until 8 months of age. D. Thalamus of rAAV-hASPA-GFP injected 3 months old mouse survived until 9 months of age. E. Hippocampus of same mouse shown in A. F. Hippocampus of same mouse shown in B. G. Enlarged lateral ventricle in an rAAV-hASPA-GFP injected mouse shown in A and E. H. Enlarged lateral ventricle in an rAAV-hASPA-GFP injected mouse shown in B and F. These studies suggest that ASPA mediated gene transfer can be one of the safest method to treat abnormal ASPA and NAA levels and sponginess seen in CD. Whether ASPA gene transfer recovers secondary effects seen in CD is yet to be studied.

Figure 6. Neuropathology of mouse brain using H&E stain. A. Thalamic vacuolization in an rAAV-GFP-injected 3 months old CD mouse survived until 6 months of age, B. survived until 5 months of age, C. survived until 8 months of age. D. Thalamus of rAAV-hASPA-GFP injected 3 months old mouse survived until 9 months of age. E. Hippocampus of same mouse shown in A. F. Hippocampus of same mouse shown in B. G. Enlarged lateral ventricle in an rAAV-hASPA-GFP injected mouse shown in A and E. H. Enlarged lateral ventricle in an rAAV-hASPA-GFP injected mouse shown in B and F. These studies suggest that ASPA mediated gene transfer can be one of the safest method to treat abnormal ASPA and NAA levels and sponginess seen in CD. Whether ASPA gene transfer recovers secondary effects seen in CD is yet to be studied.

c

HÍÉHKSÍ: ' ' î -D ^

Figure 7. Neural stem cells transplantation to the knockout mouse brain. The fluorescent cells are BrdU positive implanted cells in the (A) wild type forebrain, (B) knockout mouse forebrain, (C) wild type cerebellum and (D) knockout mouse cerebellum mouse brain. Following implantation, neural stem cells survive and migrate widely thoughout the brain and differentiate into neuronal and glial cells in an appropriate site-specific manner (Snyder et al., 1997; Na et al., 1998; Brüstle et al., 1999). Our study showed that transplantation of mouse NSCs into the knockout mouse brain survived for one month, the maximum period studied (Fig. 7) (Surendran et al., 2004b). These studies suggest that stem cell therapy is an important method to replace the lost cells in CD.

Since white matter cells are lost in CD, NSC transplantation were performed in order to test the survival and therapeutic effect of such cells.

Following implantation, neural stem cells survive and migrate widely thoughout the brain and differentiate into neuronal and glial cells in an appropriate site-specific manner (64,66,70). Our study showed that transplantation of mouse NSCs into the knockout mouse brain survived for one month, the maximum period studied (Fig. 7) (71). These studies suggest that stem cell therapy is an important method to replace the lost cells in CD.

Was this article helpful?

0 0

Post a comment