A novel system to accelerate the progression of nerve degeneration in transgenic mouse models of neuropathies

Abstract

Axon degeneration is a common hallmark of many neurodegenerative diseases. There is now an abundance of spontaneous and genetically engineered mice available to study the mechanisms of axonal degeneration and to screen for axonal protective agents. However, many of these mouse models exhibit slow progressive axonal loss which can span over many months. Consequently, there is a pressing need to accelerate the pace of axonal loss over a short interval for high-throughput screening of pharmacological and genetic therapies. Here, we present a novel technique using acrylamide, an axonal neurotoxin, to provoke rapid axonal degeneration in murine models of neuropathies. The progressive axonal loss which typically occurs over 8 months was reproduced within 7 to 10 days of the acrylamide intoxication. This approach was successfully applied to Myelin Associated Glycoprotein knockout (MAG-/-) mouse and Trembler-J mouse, a popular murine model of Charcot-Marie-Tooth disease type 1 (CMT-1). Acrylamide intoxication in transgenic mouse models offers a novel experimental approach to accelerate the rate of axonal loss over short intervals for timely in vivo studies of nerve degeneration. This report also provides for the first time an animal model for medication or toxin-induced exacerbation of pre-existing neuropathies, a phenomenon widely reported in patients with neuropathies.

Figure 1. Distal axonal degeneration in aging Trembler-J mice

(A) Gastrocnemius muscles were stained with SMI-32R (green) to detect axons and with α-bungarotoxin (red) to detect acetylcholine receptors at the muscle endplates. Muscle endplates that are positive for only α-bungarotoxin are not innervated (white arrows). NMJs in the 6-week old Tr^J mice appear compact and well defined. Furthermore there are only a few scattered denervated NMJs. In contract, most NMJs are denervated and have a diffused, irregular and fragmented appearance in the 8 month old Tr^J mice. (B) 6 week old Tr^J mice show normal appearing nerve fibers (white arrows) with minimal axonal swelling and segmentation as shown by neurofilaments (NF–H 200) immunostaining of the foot pad. In contrast, the foot pad of 8 month old Tr^J mice showed an absence of nerve fibers consistent with severe cutaneous axonal degeneration and loss. (C) This was accompanied by barely recordable CMAP responses around 1.8 mV. Examples of a typical CMAP tracing are shown in the left. (*6-week vs. 8-month old Tr^J mice, n=8, p<0.01). (D) 8-month old Tr^J mice showed severe decrease in retention time.(n=8, p<0.01). 8-month old Tr^J mice also showed a marked decline in grip strength (E) and prolonged thermal latency (F). (n=8, p<0.01).

Figure 2. Acrylamide intoxication in wild-type and Trembler-J mice

Wild-type and Trembler-J mice were exposed to 400 ppm acrylamide in drinking water for 7 days. (A) The gastrocnemius muscles were stained with SMI-32R (green) to detect axons and with α-bungarotoxin (red) to detect acetylcholine receptors at the muscle endplates. NMJs in the 6-week old wild-type (Wt) mice appear compact and well defined. There was no denervated NMJ. With acrylamide intoxication, there are a few scattered denervated NMJs. There was no significant statistically changes. (B) CMAP amplitude and (C) retention time of the Rotarod. (n=8, p<0.01) in the wild type mice even after acrylamide intoxication for 7 days. (D) In contract, in 6-week old Tr^J mice treated with 400 ppm arylamide for 7 days, most NMJs were denervated and had a diffused, irregular and fragmented appearance. (E) There were also fragmented nerve fibers (white arrows) in the foot pad consistent with severe cutaneous axonal degeneration and loss. (F) This was associated with barely recordable CMAP responses around 2 mV. (n=8, p<0.01). (G) Retention time on the Rotarod was also severely decreased. (n=8, p<0.01). (H) The grip strength likewise declined. (I) Thermal latency was markedly prolonged. (n=8, p<0.01).

Figure 3. Axonal degeneration in aging MAG−/− mice

(A) Gastrocnemius muscles were stained with SMI-32R (green) to detect axons and with α-bungarotoxin (red) to detect acetylcholine receptors at the muscle endplates. NMJs in the 6-week old MAG−/− mice appear compact and well defined. In the 8 month old MAG−/− mice, there are many denervated NMJs that have a diffused, irregular and fragmented appearance. (B) 6 week old MAG−/− mice showed normal appearing nerve fibers with minimal axonal swelling and segmentation as shown by neurofilament (NF–H 200) immunostaining (white arrows). In contrast, 8 month old MAG−/− mice showed many fragmented and enlarged nerve fibers consistent with cutaneous axonal degeneration and loss. (C) This was accompanied by a 27% reduction in CMAP responses b. representative CMAP tracing are shown in the left. (*6-week vs. 8-month old MAG−/−mice, n=8, p<0.01). (D) 8-month old AMG−/− mice also showed a decrease in retention time.(n=8, p<0.01) as well as decline in grip strength (E) and prolonged thermal latency (F). (n=8, p<0.01).

Figure 4. Acrylamide intoxication in MAG−/− mice

MAG−/− mice were exposed to 400 ppm acrylamide in drinking water for 10 days. (A) The gastrocnemius muscles were stained with SMI-32R (green) to detect axons and with α-bungarotoxin (red) to detect acetylcholine receptors at the muscle endplates. Muscle endplates that are positive for only α-bungarotoxin are not innervated (white arrows). As shown in figure 3.B, NMJs in the 6-week old MAG−/− mice appear compact and well defined. With acrylamide intoxication, there are dernervated NMJs. There were associated with reduced (B) Acrylamide intoxicated MAG−/−mice showed severe cutaneous axonal degeneration and loss as shown by neurofilament (NF–H 200) immunostaining of the foot pad. (C) There was also significant decline in CMAP amplitude and (D) retention time of the Rotarod. (n=8, p<0.01). (H) The grip strength likewise declined. (I) Thermal latency was markedly prolonged. (n=8, p<0.01). The pathologic, behavioral and electrophysiological changes associated with acrylamide treatment paralleled those seen in MAG−/− at 8 months of age.