Observations on the blood and perineurial permeability barriers of surviving nerve allografts in immunodeficient and immunosuppressed rats

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✓ The authors investigate whether there are any permeability changes in the endoneurial blood-nerve barrier and the perineurium-nerve barrier of surviving nerve allografts. In a normal nerve, the blood-nerve barrier regulates the passage of substances from endoneurial blood vessels into the endoneurium, whereas the perineurium-nerve barrier protects the endoneurium from agents that escape from permeable epineurial vessels and accumulate around the nerve. Nerves from ACI rats were transplanted into immunologically deficient nude rats or normal Fischer rats immunosuppressed with cyclosporin A. None of the nerve allografts was rejected. The blood-nerve barrier of nerve allografts at 2 and 6 weeks postoperatively was permeable to intravenously injected horseradish peroxidase, which spread into endoneurial tissue. Electron microscopy revealed that horseradish peroxidase escaped from endoneurial vessels through intercellular junctions between endothelial cells. At 24 weeks, the blood-nerve barrier of nerve allografts had recovered and the endoneurial vessels, like those in normal nerves, were impermeable to horseradish peroxidase. The perineurium-nerve barrier of nerve allografts remained impermeable to horseradish peroxidase at all times. Axons were grouped into numerous minifascicles at nerve anastomosis zones at 24 weeks. Each nerve fascicle was surrounded by an impermeable perineurium. These results demonstrate that regenerated axons in long-term surviving nerve allografts and at anastomosis zones are protected by permeability barriers. It is concluded that permeability barriers of nerve allografts are not permanently altered by a foreign environment (grafts to nude rats) even when immunosuppression with cyclosporin A is required to prevent allograft rejection (grafts to Fischer rats).

Article Information

Address for Dr. Kadota: Department of Neurosurgery, Juntendo University, Tokyo, Japan.Address reprint requests to: Andrew A. Zalewski, M.D., Laboratory of Neural Control, Building 36 Room 4A-14, National Institutes of Health, Bethesda, Maryland 20892.

© AANS, except where prohibited by US copyright law.

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    Photomicrographs illustrating the localization of horseradish peroxidase-reaction product (the black precipitate in the pictures) in cross sections of normal nerve (A–E; E is also stained with toluidine blue) and a 2-week nerve allograft (G); F is a control horseradish peroxidase section of normal nerve. A, F, and G, × 40; B, × 315; C–E, × 510. A: In normal nerve, horseradish peroxidase-reaction product is seen in the epineurium (EPI), perineurium (arrows), and lumen of an endoneurial blood vessel (ENDO). B: Although epineurial blood vessels are permeable to the horseradish peroxidase (A), the tracer spreads up to but not through the perineurium (short arrows). Note that horseradish peroxidase-reaction product is present on the luminal surface of the endothelial cells of endoneurial blood vessels (long arrows). C–E: Endoneurial vessels are not absolutely impermeable to horseradish peroxidase since horseradish peroxidase-reaction product is detectable in cells around blood vessels (C, arrow) or situated among the nerve fibers (D, arrows). The cell labeled in D can be seen after toluidine blue staining (E, arrows) to have the characteristics of a fibroblast (with an oval nucleus that extends one or more long cellular processes). F: No horseradish peroxidase-reaction product is detectable in this control nerve section from a non-horseradish peroxidase-injected rat that has been incubated in histochemical medium to detect horseradish peroxidase activity. G: Horseradish peroxidase-reaction product is present throughout the endoneurium of a 2-week degenerating nerve allograft.

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    Electron micrographs of normal nerve illustrating the localization of horseradish peroxidase in an epineurial blood vessel (A) and the perineurium (B). Epineurial blood vessels are normally permeable to intravascular horseradish peroxidase. Note the presence of horseradish peroxidase-reaction product (HRP-RP) in the junction between endothelial cells (J) and in the subendothelial basement membrane region (BM). Horseradish peroxidase-reaction product is also present in vesicles of endothelial cells (V). After its escape from epineurial blood vesels, horseradish peroxidase penetrates the outer but not inner layers of the perineurium, which consist here of several layers (labeled 1 to 7). The space between perineurial layers (labeled 1 to 7) is filled with HRP-RP. Note the absence of HRP-RP around the nerve and collagen fibers in the endoneurium (region bounded within arrows labeled “ENDO”). A, × 54,500; B, × 26,125.

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    Electron micrographs of normal nerve. A and B: Localization of horseradish peroxidase in perineurial junctions. The junction in A (outlined by arrows) is clear whereas the junction in B (outlined by arrows) is filled with horseradish peroxidase-reaction product. Perineurial junctions containing horseradish peroxidase-reaction product were rarely observed. A, × 43,600; B, × 54,500. C: A perineurium is shown in which the innermost layer (arrows) was circumferentially incomplete. No horseradish peroxidase-reaction product is present in the space between the deep perineurial layers and the endoneurium (region bounded within arrows labeled “ENDO”). × 26,125.

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    Electron micrographs of normal nerve illustrating the localization of horseradish peroxidase in an impermeable endoneurial blood vessel. Horseradish peroxidase-reaction product is present on the luminal surface of endothelial cells (arrows). Note that no horseradish peroxidase-reaction product is detectable in the junction between endothelial cells (J), the endothelial basement membrane of the blood vessel (BM), or the endoneurium (region bounded within arrows labeled “ENDO”). A, × 32,700; B, × 54,500.

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    Photomicrographs illustrating the localization of horseradish peroxidase-reaction product in a 2-week (A–D) and a 6-week (E and F) nerve allograft; D and F are also stained with toluidine blue, × 316. Endoneurial blood vessels (arrows) of 2- and 6-week grafts are now permeable to horseradish peroxidase. In some regions of nerve, horseradish peroxidase-reaction product is present focally outside permeable vessels (A) whereas in other regions the tracer spreads throughout the endoneurium (B, C, and E). Note that the perineurium in A prevents the inward movement of epineurial horseradish peroxidase, while in B the perineurium inhibits the exit of endoneurial horseradish peroxidase. Toluidine blue staining reveals that the endoneurium of a 2-week graft (D) is filled with degenerating myelin, whereas the endoneurium of a 6-week graft (F) contains numerous regenerated but thinly myelinated nerve fibers (some indicated by M in F).

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    Photomicrographs illustrating the localization of horseradish peroxidase-reaction product in 24-week nerve allografts. C and D are also stained with toluidine blue, × 315. In A, a nerve allograft from a nude rat is presented, while in B and C nerve allografts from different cyclosporin A-treated rats are illustrated; in D, a normal nerve is shown. The localization of horseradish peroxidase-reaction product is similar in nerve allografts from nude rats (A) and cyclosporin A-treated rats (B) and is comparable to that seen in normal nerve (see Fig. 1B). Note that axons in a 24-week allograft are less myelinated (C) than axons in a normal nerve (D).

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    Electron micrographs illustrating the localization of horseradish peroxidase in the endoneurium of a 6-week (A) and a 24-week (B) nerve allograft. × 14,615. A: Horseradish peroxidase-reaction product is present in the endoneurium of a 6-week graft and surrounds a regenerated and remyelinated axon. B: The endoneurium of a 24-week graft is free of horseradish peroxidase-reaction product and contains an axon with more myelin. Note the distinct outline of collagen fibers compared to those in A which are obscured by horseradish peroxidase-reaction product.

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    Electron micrographs illustrating the localization of horseradish peroxidase in a typical anastomosis zone between host and graft nerves at 24 weeks. A: Four minifascicles of nerve fibers, each surrounded by a different perineurium, are shown (arrows outline each perineurium). Note that the blood vessel is located outside the perineuria and is permeable to horseradish peroxidase. Horseradish peroxidase-reaction product (HRP-RP) in the perivascular space is prevented from reaching nearby minifascicles of nerve fibers by the outermost cellular layer of each perineurium, × 5450. B: The presence of HRP-RP is seen in the perivascular space of a blood vessel found outside a perineurium. Note that the perineurium consists of only one perineurial cell layer (arrows) which has HRP-RP on its outer but not inner surface, × 54,500.

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