The Leptin-Deficient (ob/ob) Mouse: A New Animal Model of Peripheral Neuropathy of Type 2 Diabetes and Obesity

Unless otherwise stated, all chemicals were of reagent-grade quality and were purchased from Sigma Chemical (St. Louis, MO). Rabbit polyclonal anti-nitrotyrosine antibody was purchased from Upstate (Lake Placid, NY) and mouse monoclonal anti-poly(ADP-ribose) from Trevigen (Gaithersburg, MD). Secondary Alexa Fluor 488 goat anti-rabbit and Alexa Fluor goat anti-mouse antibodies, as well as Prolong Gold Antifade Reagent, were purchased from Invitrogen (Eugene, OR). Avidin/Biotin Blocking kit, MOM Basic kit, VECTASTAIN Elite ABC kit (Standard), DAB Substrate kit, and 3,3′-diaminobenzidine were obtained from Vector Laboratories (Burlingame, CA). Rabbit polyclonal anti-protein gene product 9.5 (ubiquitin c-terminal hydrolase) antibody was purchased from Chemicon International (Temecula, CA). Other reagents for immunohistochemistry were purchased from Dako Laboratories (Santa Barbara, CA).

The experiments were performed in accordance with regulations specified by the National Institutes of Health Principles of Laboratory Animal Care (1985 revised version) and Pennington Biomedical Research Center Protocol for Animal Studies. Four-week-old C57Bl6 mice and leptin-deficient C57Bl6/J ob/ob mice were fed a standard mouse diet (PMI Nutrition International, Brentwood, MO) and had access to water ad libitum. Blood samples for glucose measurements were taken from the tail vein at the beginning of the study and the day before the animals were killed. The ob/ob mice with blood glucose >13.8 mmol/l were considered to have diabetes. The experimental groups comprised control and ob/ob mice treated with or without the AR inhibitor fidarestat (16 mg · kg⁻¹ · d⁻¹). The treatment was started at 5 weeks of age and continued for 6 weeks. The physiological and behavioral tests have been performed in the following order: tactile responses to flexible von Frey filaments (1st day), thermal algesia (2nd day), and sensory nerve conduction velocity (SNCV) and motor nerve conduction velocity (MNCV) (3rd day). Measurements of MNCV and SNCV were performed in mice anesthetized with a mixture of ketamine and xylazine (45 and 15 mg/kg body wt i.p., respectively).

The animals were sedated by CO₂ and immediately killed by cervical dislocation. One sciatic nerve from each mouse was rapidly dissected and frozen in liquid nitrogen for subsequent assessment of sorbitol pathway intermediates. Another sciatic nerve, spinal cord, DRGs, and foot pads were fixed in normal buffered 4% formalin for assessment of nitrotyrosine and poly(ADP-ribose) by immunofluorescent histochemistry.

Sciatic MNCV and hind-limb digital SNCV have been measured as we have described elsewhere (9). In all measurements, body temperature was monitored by a rectal probe and maintained at 37°C with a warming pad. Hind-limb skin temperature was also monitored by a thermistor and maintained between 36 and 38°C by radiant heat.

Tactile responses were evaluated by quantifying the withdrawal threshold of the hindpaw in response to stimulation with flexible von Frey filaments as we have described (20). The paw withdrawal latency in response to the radiant heat (15% intensity, cutoff time 30 s) was determined as we have described (20).

All sections were processed by a single investigator and evaluated blindly. Low-power observations of skin sections stained for protein gene product 9.5 were made using a Zeiss Axioskop microscope. Color images were captured with a Zeiss Axiocam HRc charged-coupled device camera at 1,300 × 1,030 resolution. Low-power images were generated with a 40× acroplan objective using the automatic capturing feature of the Zeiss Axiovision software (version 3.1.2.1). Low-power observations of sciatic nerve, spinal cord, and DRG sections stained for nitrotyrosine and poly(ADP-ribose) were made using a Zeiss Axioplan 2 imaging microscope. Color images were captured with a Photometric CoolSNAP_HQ charged-coupled device camera at 1,392 × 1,040 resolution. Low-power images were generated with a 40× acroplan objective using the RS Image 1.9.2 software.

Nitrotyrosine immunoreactivity in the sciatic nerve, spinal cord, and DRG neurons was assessed by immunofluorescent histochemistry. In brief, sections were deparaffinized in xylene, hydrated in decreasing concentrations of ethanol, and washed in water. For immunofluorescent histochemistry, rabbit polyclonal anti-nitrotyrosine antibody was used in a working dilution of 1:400. Secondary Alexa Fluor 488 goat anti-rabbit antibody was applied in a working dilution 1:200. Sections were mounted in Prolong Gold Antifade Reagent. The intensity of fluorescence was graded from 1 to 4 (1, no staining; 2, faint; 3, moderate; and 4, intense), and the immunohistochemistry score was expressed as mean ± SE for each experimental group.

Poly(ADP-ribose) immunoreactivity was assessed as described (9), with minor modifications. In brief, sections were deparaffinized in xylene, hydrated in decreasing concentrations of ethanol, and washed in water. Nonspecific binding was blocked with the mouse immunoglobulin blocking reagent supplied with the Vector MOM Basic Immunodetection kit. Then, mouse monoclonal anti–poly(ADP-ribose) antibody was diluted 1:100 in 1% BSA in Tris-buffered solution (TBS) and applied overnight at 4°C in the humidity chamber. Secondary Alexa Fluor 488 goat anti-mouse antibody was diluted 1:200 in TBS and applied for 2 h at room temperature. Sections were mounted in Prolong Gold Antifade Reagent. At the least, 10 fields of each section were examined to select 1 representative image. Representative images were microphotographed and the number of poly(ADP-ribose)–positive nuclei calculated for each microphotograph.

Intraepidermal nerve fiber density (INFD) was assessed as described (21) with minor modification. Three randomly chosen 5-μm sections from each mouse were deparaffinized in xylene, hydrated in decreasing concentrations of ethanol, and washed in water. Nonspecific binding was blocked by 10% goat serum containing 1% BSA in TBS (Dako, Carpinteria, CA) for 2 h using the Avidin/Biotin Blocking kit according to the manufacturer’s instructions. Then, rabbit polyclonal anti-protein gene product 9.5 (ubiquitin c-terminal hydrolase) antibody was applied in 1:2,000 dilution. Secondary biotinylated goat anti-rabbit IgG (H+L) antibody was applied in 1:400 dilution and the staining performed with the VECTASTAIN Elite ABC kit (Standard). For visualization of specific binding sites, the DAB Substrate kit containing 3,3-diaminobenzidine was used. Sections were counterstained with Gill’s hematoxylin, dehydrated, and mounted in Micromount mounting medium (Surgipath Medical, Richmond, IL). Intraepidermal nerve fiber profiles were counted blindly by three independent investigators, under an Olympus BX-41 microscope, and the average values were used. Microphotographs of stained sections were taken on an Axioscop 2 microscope (Zeiss) at 4× magnification, and the length of epidermis was assessed with the ImagePro 3.0 program (Media Cybernetics). Mean ± SE of the sample length (2.8 ± 0.3 mm) was investigated to calculate a number of nerve fiber profiles per millimeter of epidermis.

Glucose, sorbitol, and fructose concentrations in sciatic nerve have been spectrofluorometrically assessed by enzymatic procedures with hexokinase/glucose 6-phosphate dehydrogenase, sorbitol dehydrogenase, and fructose dehydrogenase, as we described elsewhere (22).

The results are expressed as means ± SE. Data were subjected to equality of variance F test and then to log transformation, if necessary, before one-way ANOVA. Where overall significance (P < 0.05) was attained, individual between-group comparisons were made using the Student-Newman-Keuls multiple range test. Significance was defined at a P value <0.05. When between-group variance differences could not be normalized by log transformation (datasets for body weights and plasma glucose), the data were analyzed by the nonparametric Kruskal-Wallis one-way ANOVA, followed by the Bonferroni/Dunn test for multiple comparisons.

Both initial and final body weights and initial and final blood glucose concentrations were higher in ob/ob mice compared with nondiabetic controls (Table 1). Fidarestat did not affect weight gain or blood glucose concentrations in either nondiabetic control or ob/ob mice.

Sciatic nerve glucose, sorbitol, and fructose concentrations were increased in ob/ob mice compared with controls (Table 2). Fidarestat treatment did not affect sciatic nerve glucose concentrations but essentially prevented an increase in sorbitol and fructose concentrations in ob/ob mice. Sciatic MNCV was 16% lower in untreated ob/ob mice compared with nondiabetic controls (Fig. 1A). In contrast, fidarestat-treated ob/ob mice preserved normal MNCV. In a similar fashion, hind-limb digital SNCV was reduced by 22% in ob/ob mice compared with nondiabetic controls (Fig. 1B), and this reduction was essentially prevented by fidarestat. Fidarestat did not affect MNCV or SNCV in nondiabetic control mice.

The latency of hind-paw withdrawal in response to radiant heat was increased by 88% in ob/ob mice compared with nondiabetic controls (P < 0.01), which is consistent with clearly manifest thermal hypoalgesia (Fig. 2A). Fidarestat partially (to 131% of control value, P < 0.01 vs. nondiabetic controls and P < 0.01 vs. untreated diabetic group) prevented a diabetes-induced increase in paw withdrawal latency. Of interest, paw withdrawal latency in nondiabetic control animals treated with fidarestat was 32% higher than in the corresponding untreated group.

Another sensory abnormality developing in ob/ob mice was tactile allodynia. Tactile withdrawal threshold in response to light touch with flexible von Frey filaments was reduced 8.2-fold in ob/ob mice compared with nondiabetic controls (P < 0.01; Fig. 2B). Tactile response thresholds were 1.7-fold higher in ob/ob mice treated with fidarestat compared with the corresponding untreated group; however, the difference between fidarestat-treated and untreated ob/ob mice did not achieve statistical significance (P = 0.44). Furthermore, tactile response thresholds remained 4.9-fold lower in fidarestat-treated ob/ob mice compared with nondiabetic controls (P < 0.01). Fidarestat did not affect tactile response thresholds in the nondiabetic control group.

INFD was reduced by 78% in ob/ob mice compared with nondiabetic controls (P < 0.01; Fig. 3). Fidarestat essentially (to 72% of control value, P < 0.01 vs. untreated ob/ob mice and P = 0.06 vs. nondiabetic controls) prevented intraepidermal nerve fiber loss in ob/ob mice, without affecting this variable in control mice.

Nitrotyrosine immunoreactivity was increased by 78% in the sciatic nerve of ob/ob mice compared with nondiabetic controls (Fig. 4A and B), and this increase was prevented by fidarestat treatment. Fidarestat did not affect nitrotyrosine immunoreactivity in nondiabetic control mice. The number of the sciatic nerve poly(ADP-ribose)–positive nuclei was approximately twofold greater in ob/ob mice compared with nondiabetic controls (P < 0.01; Fig. 4C and D). No significant differences in the numbers of sciatic nerve poly(ADP-ribose)–positive nuclei were found between fidarestat-treated ob/ob mice and nondiabetic controls. Fidarestat did not affect poly(ADP-ribose) immunoreactivity in nondiabetic control mice. The similar patterns were observed in the spinal cord (Fig. 5) and DRGs (Fig. 6). In both sites, nitrotyrosine and poly(ADP-ribose) immunoreactivities were increased in ob/ob mice compared with nondiabetic controls (P < 0.01 for all comparisons). No significant differences in the spinal cord or DRG nitrotyrosine immunoreactivities or the numbers of poly(ADP-ribose)–positive nuclei were found between fidarestat-treated ob/ob mice and nondiabetic controls. Fidarestat did not affect nitrotyrosine or poly(ADP-ribose) immunoreactivities in the spinal cord and DRGs of nondiabetic control mice.

Recent clinical studies suggest that subjects with impaired glucose tolerance and obesity have increased risk for developing motor and sensory nerve conduction slowing and small sensory nerve fiber neuropathy (23–26). Obesity is clearly associated with increased prevalence of neuropathy in overt type 1 and type 2 diabetes (27,28). Pathogenetic mechanisms underlying PDN developing in the absence of fasting hyperglycemia (pre-diabetes) or under conditions of mild hyperglycemia in type 2 diabetes are poorly studied. Neurological and neurovascular deficits associated with type 2 diabetes have been described in sucrose-fed Otsuka Long-Evans Tokushima fatty (OLETF) rats (14), Goto-Kakizaki and BBZDR/Wor rats (15,16), and Zucker obese and Zucker diabetic fatty rats (12,13). Our findings indicate that leptin-deficient (ob/ob) mice with moderate hyperglycemia, hyperinsulinemia, and obesity (17,18) develop MNCV and SNCV deficits, thermal hypoalgesia, tactile allodynia, and epidermal sensory fiber loss, which are characteristic of human subjects with diabetes, and thus represent a valuable animal model of PDN of type 2 diabetes and obesity. This model has a number of advantages over existing animal models of type 2 diabetic neuropathy mentioned above. Whereas ob/ob mice have clearly manifest thermal hypoalgesia, i.e., condition observed in human subjects with PDN (3), BBZDR/Wor rats (15) and Zucker diabetic fatty rats (13) have persistent thermal hyperalgesia, which is a transient phenomenon in PDN in humans (29). In addition, blood glucose concentrations often achieve a very high level, ∼30 mmol/l, in Zucker diabetic fatty rats (13), which makes the pathological condition in this model very different from type 2 diabetes. In contrast, nonfasting blood glucose concentrations in ob/ob mice typically do not exceed 20 mmol/l. OLETF rats develop nerve conduction deficit and peripheral nerve sorbitol pathway intermediate accumulation only after an extended (∼8 weeks) feeding of sucrose to achieve severe hyperglycemia (14). In contrast, ob/ob mice developed clearly manifest motor and sensory nerve conduction slowing and peripheral nerve sorbitol pathway intermediate accumulation when fed a regular mouse diet to maintain moderate hyperglycemia. Goto-Kakizaki and Zucker obese rats better suit the experimental studies of neuropathy of pre-diabetes rather than of type 2 diabetes, as both models either do not develop overt fasting hyperglycemia (Goto-Kakizaki rats [15]) or develop it very late (∼40 weeks of age) after a decline in β-cell function (Zucker obese rats [12]).

The severities of MNCV and SNCV deficits and thermal hypoalgesia in ob/ob mice were quite similar to those demonstrated in rats and mice with type 1 (streptozotocin- induced) diabetes (9,30 and I.G.O., V.V.L., unpublished observations). In addition, ob/ob mice clearly developed manifest tactile allodynia, a condition in which a light touch is perceived as painful. This phenomenon is observed in a considerable proportion (from 30% [31] to 47% [26]) of human subjects with diabetes and, so far, has not been described in mouse models of PDN. The severity of allodynia as determined by reduction in threshold of response to light touch with flexible von Frey filaments is quite comparable in 11-week-old ob/ob mice and rats with an 8-week duration of streptozotocin-induced diabetes (30).

Historically, small-caliber nerve fiber degeneration has not been evaluated in either human subjects with diabetes or animal models due to a lack of objective measures. Emerging new techniques such as corneal confocal microscopy, a reiterative, rapid, noninvasive in vivo imaging technique for quantitative assessment of degeneration and regeneration of corneal nerve fibers (32), and skin biopsy with visualization and quantitation of epidermal nerve fibers (21,33,34) generated enormous potential for better understanding the mechanisms underlying small nerve fiber degeneration, a phenomenon that may ultimately lead to a complete loss of sensory function and is a major cause of foot amputation. Recent studies revealed that subjects with both type 1 and type 2 diabetes, but not those with metabolic syndrome, display epidermal nerve fiber loss (33–37). In our study, 11-week-old ob/ob mice developed a dramatic (78%) reduction in INFD compared with age-matched nondiabetic controls. Such a dramatic loss of epidermal nerve fibers probably reflects a synergistic effect of hyperglycemia and obesity. Note, like human subjects with metabolic syndrome (37), high-fat–fed mice with impaired glucose tolerance and obesity, but without overt hyperglycemia, do not display any epidermal fiber loss (I.G.O., V.R.D., V.V.L., unpublished observations). Moreover, a severe reduction in INFD in ob/ob mice exceeded that observed in streptozotocin-induced diabetic mice with an 8- to 12-week duration of diabetes and had more severe hyperglycemia but no obesity (∼50%) (V.R.D., V.V.L., I.G.O., unpublished observations).

In evaluating amenability of PDN in ob/ob mice to pathogenetic treatment, we have chosen the AR inhibitor fidarestat for the following reasons. First, ob/ob mice display evidence of increased sorbitol pathway activity in the peripheral nerve. Second, numerous findings suggest that increased AR activity is the major “upstream” mechanism that accounts for or provides the important contribution to numerous diabetes-induced abnormalities in peripheral nervous system and other tissue sites for diabetes complications. During the last 20 years, increased AR activity was found to result in myo-inositol depletion and decrease in Na⁺/K⁺ ATP-ase activity (38), NAD⁺/NADH redox changes (39), alterations in arachidonic acid metabolism (40), depletion of nerve growth factor and nerve growth factor–regulated neuropeptides (41,42), as well as ciliary neurotrophic factor (43), oxidative-nitrosative stress (44), and, recently, activation of mitogen-activated protein kinases (45), poly(ADP-ribose) polymerase (44), and cyclooxygenase-2 (K. Ramos, N. Calcutt, unpublished observations). Third, AR inhibition or gene deficiency resulted in prevention and correction of functional and morphological abnormalities of PDN in streptozotocin-induced diabetic rats and mice (4,5,19,39,46). Furthermore, several clinical trials with different AR inhibitors revealed that robust inhibition of the enzyme activity is accompanied by a statistically significant increase in nerve conduction velocity and myelinated fiber density, as well as improvement of subjective symptoms of PDN (including numbness, spontaneous pain, sensation of rigidity, paresthesia in the sole upon walking, heaviness in the foot, and hypesthesia) (47–50). In the present study, AR inhibition with fidarestat resulted in complete or partial prevention of peripheral nerve sorbitol pathway intermediate accumulation, motor and sensory nerve conduction slowing, and thermal hypoalgesia in 11-week-old ob/ob mice. Furthermore, we obtained the first evidence of the efficacy of an AR inhibitor treatment in preventing small sensory nerve fiber degeneration characteristic for PDN and maintaining normal INFD. Note that beneficial effects of fidarestat on INFD in ob/ob mice were consistent with alleviation of thermal hypoalgesia, a functional correlate of small sensory nerve fiber degeneration. Alleviation of thermal hypoalgesia with the AR inhibitor fidarestat treatment is in line with previous studies with other AR inhibitors in the streptozotocin-induced diabetic rat model (30). Furthermore, alleviation of sensory neuropathy and, in particular, intraepidermal nerve fiber loss in our experimental study is consistent with improvement of subjective symptoms of PDN, i.e., numbness, sensation of rigidity, heaviness in the foot, and others, in the fidarestat clinical trial (48). Of interest, whereas tactile allodynia in streptozotocin-induced diabetic rats was totally nonresponsive to an AR inhibitor treatment (30), tactile response thresholds in fidarestat-treated ob/ob mice were ∼1.7-fold higher than in the corresponding untreated group. This difference, however, was minor compared with an approximate eightfold increase in tactile sensitivity in ob/ob mice compared with nondiabetic controls. The biochemical mechanisms underlying tactile allodynia are not understood; of interest, in the streptozotocin-induced diabetic rat model, poly(ADP-ribose) polymerase inhibition (20), but not AR inhibition (30), resulted in partial restoration of normal sensitivity to light touch. Perhaps a better understanding of diabetes-induced allodynia may be achieved by utilizing both functional (tactile response thresholds) and anatomical (Meissner corpuscle counts at the dermal-to-epidermal junction) assessment of this condition in PDN-related studies, including those addressing the role for AR. Of note, recent experimental (51,52) and clinical studies (50) have demonstrated 1) the important role for AR in pathological conditions, others than diabetes, and 2) the best efficacy of an AR inhibitor treatment in subjects with good glycemic control. Thus, current understanding of the metabolic function of sorbitol pathway is incomplete, which impedes the progress in identifying its role in disease, including PDN.

Evidence for an important role of free radicals and oxidants in PDN (9,10,53,54), including PDN in humans (55), is emerging. Our findings indicate that ob/ob mice have enhanced oxidative-nitrosative stress, manifest by increased nitrotyrosine and poly(ADP-ribose) immunoreactivities in peripheral nerve, spinal cord, and DRGs. An AR inhibitor treatment prevented nitrotyrosine and poly(ADP-ribose) accumulation in all three tissue sites of PDN. The latter is consistent with 1) AR localization in rodent peripheral nerve, spinal cord, and DRGs (56,57) and 2) numerous findings (rev. in [44]) indicating that increased AR activity is localized upstream consequent to oxidative-nitrosative stress and poly(ADP-ribose) polymerase activation in tissue sites for diabetes complications.

In conclusion, the leptin-deficient ob/ob mouse is a new animal model of neuropathy of type 2 diabetes and obesity that develops both large motor and sensory fiber and small sensory fiber PDN, offers a number of advantages over existing animal models, and responds to pathogenetic treatment. The results support the role for increased AR activity in functional and structural changes of PDN in type 2 diabetes.

This study was supported by an American Diabetes Association research grant and a Sanwa Kagaku Kenkyusho grant (both to I.G.O.).