Neuroimmunophilin ligands protect cavernous nerves after crush injury in the rat: new experimental paradigms

Abstract

Objectives: We investigated the effects of the orally bioavailable non-immunosuppressive immunophilin ligand GPI 1046 (GPI) on erectile function and cavernous nerve (CN) histology following unilateral or bilateral crush injury (UCI, BCI, respectively) of the CNs.

Methods: Adult male Sprague-Dawley rats were administered GPI 15 mg/kg intraperitoneally (ip) or 30 mg/kg orally (po), FK506 1 mg/kg, ip, or vehicle controls for each route of administration just prior to UCI or BCI and daily up to 7 d following injury. At day 1 or 7 of treatment, erectile function induced by CN electrical stimulation was measured, and electron microscopic analysis of the injured CN was performed.

Results: Intraperitoneal administration of GPI to rats with injured CN protected erectile function, in a fashion similar to the prototypic immunophilin ligand FK506, compared with vehicle-treated animals (93%+/-9% vs. 70%+/-5% vs. 45%+/-1%, p<0.01, respectively). Oral administration of GPI elicited the same level of significant protection from CN injury. GPI administered po at 30 mg/kg/d, dosing either once daily or four times daily with 7.5 mg/kg, provided nearly complete protection of erectile function. In a more severe BCI model, po administration of GPI maintained erectile function at 24 h after CN injury. Ultrastructural analysis of injured CNs indicated that GPI administered at the time of CN injury prevents degeneration of about 83% of the unmyelinated axons at 7 d after CN injury.

Conclusions: The orally administered immunophilin ligand GPI neuroprotects CNs and maintains erectile function in rats under various conditions of CN crush injury.

Fig. 1

ICP is maintained in UCI following intraperitoneal treatment of GPI. The right CN was crushed as described in methods. Both vehicle and GPI (15 mg/kg, ip) treatment preceded the nerve crush by 30 min. Twenty-four hours later, ICP was measured during a 60-s electrical stimulation of the left CN (control) and the right CN (crush). (A). The ICP responses were recorded as a function of time and depicted for both the GPI-treated (left panel) and the vehicle-treated (right panel) rats. (B). Comparison of the maximum ICP response data from graphs in A. Statistical difference defined as * p < 0.01, n = 6 per group. CN = cavernous nerve; GPI = GPI-1046; ICP = intracavernosal pressure; ip = intraperitoneal; UCI = unilateral crush injury.

Fig. 2

Intraperitoneal administration of FK506 and GPI maintains ICP responses in rats with UCI. Rats were treated with vehicle, GPI (15 mg/kg), or FK506 (1 mg/kg) for 30 min prior to UCI. Twenty-four hours later, ICP was measured during a 60-s electrical stimulation of the left CN (control) and the right CN (crush). The ICP response was expressed as %ICP of the uninjured CN stimulation for UCI. Statistical difference defined as * p < 0.05, ** p < 0.01, n = 6 per group. See Figure 1 for definitions of abbreviations.

Fig. 3

Oral administration of GPI maintains ICP responses in rats with UCI. Both the vehicle and GPI (30 mg/kg) treatment preceded the nerve crush by 30 min. Twenty-four hours later, ICP was measured during a 60-s electrical stimulation of the left CN (control) and the right CN (crush). Data were expressed as mean ± SE of maximum ICP. Statistical difference defined as * p < 0.05, n = 8 per group. See Figure 1 for definitions of abbreviations.

Fig. 4

Oral administration of GPI maintains ICP responses in rats 7 d after UCI. Rats were treated for 7 d with vehicle or GPI starting 30 min prior to UCI. (A) Vehicle or GPI (30 mg/kg/d). (B) Vehicle or GPI (7.5 mg/kg, po). Seven days after UCI, ICP was measured during a 60-s electrical stimulation of the left CN (control) and the right CN (crush). The ICP response was expressed as %ICP of the uninjured CN stimulation. Statistical difference defined as ** p < 0.01, n = 8/group. See Figure 1 for definitions of abbreviations.

Fig. 5

Oral administration of GPI maintains ICP responses in rats with BCI. Rats were administered vehicle or GPI (30 mg/kg) 30 min prior to BCI. A group of sham rats were run in parallel for comparison. Twenty-four hours later, ICP was measured during a 60-s electrical stimulation of the left CN (control) and the right CN (crush). Data was expressed as mean ± SE of maximum ICP. Statistical difference defined as ** p < 0.001, n = 8 per group. BCI = bilateral crush injury; see Figure 1 for definitions of other abbreviations.

Fig. 6

GPI treatment prevents degeneration of unmyelinated axons in the distal CN. Rats with UCI were treated with vehicle or GPI (15 mg/kg, ip), and the CNs were evaluated for axonal degeneration by EM (see Methods). (A) Quantitation of the right CN (crush) unmyelinated axon survival at 7 d after injury in vehicle- or GPI- treated animals (N = 4 per group, p = 0.007). Representative EMs of (B) left CN (control); (C) right CN (crush) with GPI treatment; (D) right CN (crush) with vehicle treatment. There are more degenerating unmyelinated axons in the vehicle-treated nerves (narrow arrows point to some of the degenerating axons; thick arrow points to a degenerating small myelinated axon). EM = electron microscopy; see Figure 1 for definitions of other abbreviations.