Validating indicators of CNS disorders in a swine model of neurological disease

Abstract

Genetically modified swine disease models are becoming increasingly important for studying molecular, physiological and pathological characteristics of human disorders. Given the limited history of these model systems, there remains a great need for proven molecular reagents in swine tissue. Here, to provide a resource for neurological models of disease, we validated antibodies by immunohistochemistry for use in examining central nervous system (CNS) markers in a recently developed miniswine model of neurofibromatosis type 1 (NF1). NF1 is an autosomal dominant tumor predisposition disorder stemming from mutations in NF1, a gene that encodes the Ras-GTPase activating protein neurofibromin. Patients classically present with benign neurofibromas throughout their bodies and can also present with neurological associated symptoms such as chronic pain, cognitive impairment, and behavioral abnormalities. As validated antibodies for immunohistochemistry applications are particularly difficult to find for swine models of neurological disease, we present immunostaining validation of antibodies implicated in glial inflammation (CD68), oligodendrocyte development (NG2, O4 and Olig2), and neuron differentiation and neurotransmission (doublecortin, GAD67, and tyrosine hydroxylase) by examining cellular localization and brain region specificity. Additionally, we confirm the utility of anti-GFAP, anti-Iba1, and anti-MBP antibodies, previously validated in swine, by testing their immunoreactivity across multiple brain regions in mutant NF1 samples. These immunostaining protocols for CNS markers provide a useful resource to the scientific community, furthering the utility of genetically modified miniswine for translational and clinical applications.

Fig 1. Immunolabeling of known markers in various regions of NF1 mutant miniswine brain.

Brain regions are identified as cortex (CTX), cerebellum (CB), thalamus (THAL), and hippocampus (HPC). A-D: GFAP+ expression in the cortex, cerebellum, and thalamus, localized to the membrane and filaments of astroyctes. Arrows indicate classic, star-shaped morphology of astrocytes. E-H: Iba1+ expression in the cortex, cerebellum, and thalamus, localized to the cytoplasm of microglia. Arrows indicate both ramified and ameboid-like microglia. Scale bar: 50μm. I-L: MBP+ expression in the cortex, cerebellum, and hippocampus, localized to the membrane and cytoplasm of mature oligodendrocytes. Scale bar: 500μm. I’-L’: Enhanced magnification of panels I-L. Scale bar: 200μm. Asterisks indicate lack of immunolabeling in neurons, as expected.

Fig 2. Immunolabeling of microglial and pre-oligodendrocyte markers in the NF1 mutant and wild-type miniswine brain.

Brain regions are identified as cortex (CTX), cerebellum (CB), thalamus (THAL), hippocampus (HPC) and cerebral aqueduct (CGG). A-D: CD68+ expression in the cortex, cerebellum, hippocampus, and thalamus, localized to the membrane of microglia. Arrows indicate ameboid-like microglia. E: CD68+ expression in mouse cerebral cortex. F-I: NG2+ expression in the cortex, cerebellum, and thalamus, localized to the membrane and cytoplasm of oligodendrocyte progenitors. Arrows indicate immature oligodendrocytes. J: NG2+ expression in mouse cerebral cortex. K-N: O4+ expression in the cortex, cerebellum, cerebral aqueduct, and thalamus localized to the membrane of pre-oligodendrocytes. Arrows indicate pre-oligodendrocytes. O: O4+ expression in mouse cerebral cortex. Asterisks indicate lack of immunolabeling. Scale bar: 50μm. WT animals shown as a healthy control.

Fig 3. Immunolabeling of mature oligodendrocyte lineage markers in the NF1 mutant and wild-type miniswine brain.

Brain regions are identified as cortex (CTX), cerebellum (CB), cerebral aqueduct (CGG) and corpus callosum (CC). A-D: Olig2+ expression in the cortex, cerebellum, cerebral aqueduct, and thalamus in wild-type, localized to the nucleus and cytoplasm of oligodendrocytes. Arrows indicate mature oligodendrocytes. E: Olig2+ expression in mouse cerebral cortex. F-I: Myelin PLP+ expression in the cortex, cerebellum, corpus callosum, and thalamus in wild-type localized to the membrane and cytoplasm of multiple cells as well as to the filamentous structures of the neuropil. Non-specific staining to neurons as well as background staining was found. J: Myelin PLP+ expression in mouse cerebral cortex. Asterisks indicate lack of immunolabeling. WT animals shown as a healthy control. Scale bar: 50μm.

Fig 4. Immunolabeling of neuron markers in the NF1 mutant and wild-type miniswine brain.

Brain regions are identified as cortex (CTX), cerebellum (CB), hippocampus (HPC) and cerebral aqueduct (CGG). A-D: Doublecortin+ expression in the cortex, cerebellum, hippocampus, and cerebral aqueduct in wild-type miniswine, localized to the cytoplasm of neurons. Arrows indicate immature neurons, with minimal staining in the cerebellum. E: Doublecortin+ expression in mouse cerebral cortex. F-I: GAD67+ expression in the cortex, cerebellum, hippocampus, and thalamus in wild-type localized to the cytoplasm of neurons. Arrows indicate GABAergic neuron cell bodies. J: GAD67+ expression in mouse cerebral cortex. K-N: Tyrosine H+ expression in the cortex, cerebellum, and cerebral aqueduct in NF1 and wild-type miniswine, localized to the cytoplasm of neurons. Arrows indicate dopaminergic neurons. O: Tyrosine H+ expression in mouse cerebral cortex. Asterisks indicate lack of immunolabeling. Scale bar: 50μm.

Fig 5. Immunolabeling miniswine DRGs.

A-B: Miniswine DRGs were immunostained with commercial antibodies against TRPV1 and CGRP. C-D: Negative controls omitting primary antibodies. Nuclei were counterstained with DAPI. Scale bars: 50μm.