Administration of an aldose reductase inhibitor, ONO-2235, to streptozotocin-diabetic mice restores reductions of DRG neuronal attachment to extracellular matrix proteins in vitro

Kazunori Sangoa,*, Hidenori Horieb, Shuji Inouea
aDivision of Geriatric Health and Nutrition, National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan
bDepartment of Physiology, Yokohama City University, School of Medicine, Yokohama 236-0004, Japan

Received 16 December 1998; received in revised form 2 February 1999; accepted 2 February 1999

Abstract

Attachments of cultured dorsal root ganglion (DRG) neurons to the extracellular matrix (ECM) proteins (type I and IV collagens, laminin and fibronectin) and the adhesion ligand arginine-glycine-aspartic acid (RGD) were impaired in mice 2 weeks after the induction of diabetes by streptozotocin (STZ). However, administration of the aldose reductase inhibitor, ONO-2235, to the STZ- diabetic mice for 1 week restored DRG neuronal attachment to the ECM proteins and RGD to a level close to normal mice. These results suggest that activation of the aldose reductase and subsequent metabolic disorders in diabetic animals may play an important role in detrimental alterations of the neuronal cell-surface receptors for the ECM proteins.  1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Diabetic neuropathy; Dorsal root ganglion neurons; In vitro; Attachment; Extracellular matrix proteins; Aldose reduc- tase inhibitor

Diabetic neuropathy is one of the most common compli- cations of diabetes mellitus. Although its pathogenesis remains unclear, metabolic disorders due to insulin defi- ciency and hyperglycemia are considered to be crucial for its development [15,19]. One of the most important initiat- ing abnormalities responsible for diabetic neuropathy is believed to be the activation of the aldose reductase (AR). AR is the first enzyme in the polyol pathway and catalyzes the reduction of glucose to sorbitol, which in turn can be oxidized by the enzyme sorbitol dehydrogenase (SDH) to fructose. The activation of AR induced by hyperglycemia dramatically enhances the flux through the polyol pathway, and this enhancement may directly or indirectly affect nerve functions, thereby leading to the development of neuropathy [15,17,19]. This concept has been supported by a number of studies in which aldose reductase inhibitors (ARIs) have ameliorated functional, biochemical, and structural abnorm- alities in the peripheral nerve in experimental diabetic ani-

* Corresponding author. Tel.: +81-3-3203-5723., fax: +81-3-3205- 9536; e-mail: [email protected]
mals [2,8,9,14,18]. However, clinical trials of ARIs have not yet proven the significant efficacy of ARIs for patients with diabetes of relatively long duration [4,11,17], suggesting that factors other than the activation of AR (i.e. non-enzy- matic glycation of nerve proteins, endoneurial hypoxia, etc.) may contribute to the pathological progression in diabetic neuropathy [1,7].
We have applied the culture method of dorsal root gang- lion (DRG) neurons to experimental diabetic mice to study diabetic neuropathy in vitro [12,13]. DRG neurons are con- stituents of the sensory system, which is most vulnerable in diabetic neuropathy, and this in vitro system has allowed us to investigate the initial functional disorders of these neu- rons exposed to hyperglycemia in vivo. We have previously reported that attachments of cultured DRG neurons to the extracellular matrix (ECM) proteins such as types I and IV collagens, laminin and fibronectin were impaired in diabetic mice 6 weeks after the injection of streptozotocin (STZ) [12]. In the present study, we examined whether the ability of DRG neurons to bind to the ECM proteins is affected at an earlier stage of diabetes (2 weeks after the STZ injec-

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158 K. Sango et al. / Neuroscience Letters 263 (1999) 157–160

tion). We also observed the effects of an ARI on the dia- betes-induced changes of DRG neuronal attachment.
Three-month-old female C57BL mice were injected intraperitoneally with 200 mg/kg STZ (Sigma) dissolved in 0.4 ml sodium citrate buffer (pH 4.5). Age-matched nor- mal control animals were injected with sodium citrate buffer alone. One week after the injection, blood glucose levels were measured by glucose oxidase method. The STZ- injected mice with blood glucose concentrations of more than 16.7 mM (300 mg/dl) were considered to be diabetic. Then the mice were divided into three groups (1) control group, (2) untreated diabetic group and (3) ARI-treated dia- betic group. In the last group, diabetic mice were treated with an aldose reductase inhibitor, ONO-2235 (Epalrestat; Ono Pharmaceutical Company, Japan) [4]. ONO-2235 was suspended in 0.5% carboxymethylcellulose and given at a daily dose of 20 mg/kg body weight by gastric intubation for 1 week. The control and untreated diabetic groups were only administered 0.5% carboxymethylcellulose solution. Blood glucose concentrations of the mice one week after the administration were 150.3  34.1 mg/dl in the control group, 416.7  110.0 mg/dl in the untreated diabetic group, and 421.0  96.9 mg/dl in the ARI-treated diabetic group, respectively (each group contained four individuals); ONO-2235 did not affect the blood glucose levels in dia- betic mice.
The primary cultures of adult mouse DRG neurons were performed as we previously reported [12]. Briefly, the mice in each group were sacrificed by ether exposure. From 35 to 40 ganglia (from the thoracic to sacral levels) were dis- sected from each animal and enzymatically dissociated with collagenase and trypsin. The dissociated tissues were then layered on 30% Percoll (Pharmacia) solution and sub- jected to density gradient centrifugation (5 min, 200  g) to eliminate the myelin sheath and non-neuronal cells. This procedure resulted in a yield of more than 5  104 neurons with high purity per mouse in each group.
Thirty-five millimeter Falcon culture dishes were coated with the following substrates: (1) poly-l-lysine (Sigma, 10 mg/ml) (PL); (2) poly-l-lysine and type I collagen (from rat tail, 30 mg/ml) (PL  CL-I); (3) poly-l-lysine and type IV
collagen (Collaborative Res. Inc., 30 mg/ml) (PL  CL-IV);
⦁ poly-l-lysine and laminin (Collaborative Res. Inc., 20 mg/ml) (PL  LM); (5) poly-l-lysine and fibronectin (Col- laborative Res. Inc., 30 mg/ml) (PL  FN); (6) arginine-gly- cine-aspartic acid (Collaborative Res. Inc., 30 mg/ml) (RGD).
Each of CL-I, CL-IV, LM and FN was added to the sur- face of the PL-coated dishes. DRG neurons dissociated from each mouse were suspended in a serum-free medium (SFM), and seeded onto the culture dishes coated with the above six substrates. Areas of 5  5 mm2 were delineated by lines on the bottom of these dishes prior to seeding cells, and cell density was adjusted to approximately 750 cells within this area (3.0  103/cm2) of each dish. Thirty minutes after incubation of the cells in a humidified atmosphere contain- ing 5% CO2 at 37C, the number of DRG neurons within the designated area of each dish was counted under a phase- contrast light microscope. The cells were kept in SFM and allowed to adhere to the substrate for 6 h, then washed three times with SFM to exclude non-attached cells and the num- ber of viable cells were counted under the microscope. Cell viability was determined by Trypan blue exclusion. The ratio (%) of cells attached to the substrate in each culture dish was expressed as B/A (A: number of cells measured 30 min after seeding, B: number of cells measured after wash- ing with SFM after 6 h of culture) [12].
Dissociated cell culture and subsequent cell attachment assay was performed in individual animals. The attachment assay in each substrate was repeated between three to seven times, with four animals in each group. Means and standard deviations were calculated and analyzed according to the unpaired Student’s t-test; P-values of < 0.01 were consid- ered significant.
Table 1 shows the ratios of cell attachment to each sub- strate in the control, untreated diabetic, and ARI-treated diabetic groups after 6 h in culture. There were no signifi- cant differences in the ratios of attachment to PL alone among the three animal groups. In the control group, addi- tion of CL-I, CL-IV, LM, or FN to PL significantly increased the average ratio of attachment from 71.9% to nearly 90%. In the untreated diabetic group, however, coat-

Table 1
Ratios of attachment of DRG neurons to the extracellular substrates after 6 h in culture in control, untreated diabetic and ARI-treated diabetic mice

Percentage of cells attached
Substrate Control Untreated diabetic ARI-treated diabetic
poly-L-lysine(PL) 71.9  4.0 70.0  10.4 70.6  10.8
PL  type l collagen 88.6  1.8*,** 66.3  7.8 80.2  3.6
PL  type IV collagen 87.2  5.2*,** 65.1  4.8 82.0  10.5**
PL  laminin 86.4  5.3*,** 66.4  8.5 85.3  7.6**
PL  fibronectin 85.9  3.7*,** 71.0  7.1 83.8  6.2**
Arg-Gly-Asp(RGD) 80.4  3.7** 65.0  2.9 77.9  2.6**
Dissociated cell culture and subsequent cell attachment assay was performed in individual animals. The values represent the mean and standard deviations of three to seven experiments with four animals in each group ( indicates standard deviations).
*P < 0.01 compared with PL in the control group. **P < 0.01 compared with the corresponding value in the diabetic group.

K. Sango et al. / Neuroscience Letters 263 (1999) 157–160 159

ing of none of these four substrates onto PL enhanced the ratio of attachment. RGD, an adhesion site in the ECM proteins, is believed to be essential for binding to many integrin receptors [3,5]. The average ratio of attachment to RGD was also significantly lower in the untreated diabetic group (average 65.0%) than that in the control group (80.4%). On the other hand, the ratios of attachment to these five substrates in the ARI-treated diabetic group were significantly higher than those in the untreated diabetic group and reached a level close to those in the controls.
As found in the previous studies [12,20], the ECM pro- teins added to PL enhanced attachment of normal mouse DRG neurons in vitro in the present study. This enhance- ment is known to result from specific bindings of neuronal cell-surface receptors (i.e. integrins) to the ligands (i.e. RGD) on the ECM proteins [3,5]. However, none of the ECM proteins improved DRG neuronal attachment in the untreated diabetic mice. This result indicates that hypergly- cemia in animals induced a decrease in quantity and/or quality of the cell-surface receptors, which enable DRG neurons to bind to the ligands of these proteins. The period of the disease in which the neurons are exposed to hyper- glycemia in animals is one of the most important factors in evaluating the impaired attachment of diabetic neurons in vitro. We previously reported this impairment in mice with STZ-diabetes for 6 weeks [12]. In the present study, a decline of the neuronal attachment to the ECM proteins and the RGD ligand was observed in a stage of diabetes as early as 2 weeks after the STZ-injection.
This study also demonstrated that administration of the aldose reductase inhibitor, ONO-2235, to STZ-diabetic mice for a week restored the reductions of DRG neuronal attachment to the ECM proteins and RGD in vitro. Since ONO-2235 had no effect on blood glucose levels in the diabetic mice, it is plausible that the activation of AR due to hyperglycemia, rather than hyperglycemia itself, can be crucial for the impairment of the neuron-matrix binding.
The activation of AR enhances the flux through the polyol pathway, which is believed to cause abnormalities of nerve myo-inositol, phosphoinositide and Na -K -ATPase [15,17]. These molecules are essential for neural cell mem- brane functions, and thus, metabolic disorders of these molecules might impair neural cell membranes in diabetic animals. Such membrane impairments can alter the struc- ture and function of the cell-surface receptors, since the receptors are composed of transmembrane proteins embedded in a lipid bilayer [3,5]. However, Llewelyn et al. reported that accumulation of sorbitol and fructose in the diabetic mouse nerves were less marked than those in diabetic rat nerves [6]. It is also addressed that a deficit in motor nerve conduction velocity (MNCV) of diabetic mutant mice was significantly prevented by aldose reduc- tase inhibition without affecting nerve myo-inositol content [8], and that levels of sorbitol and fructose in sciatic nerve of STZ-diabetic mice did not correlate with the degree of MNCV deficit [10]. These reports suggest that activation

of the polyol pathway itself is not so much responsible for the development of neuropathy in diabetic mice. Therefore, other metabolic disorders due to the activation of AR, than the enhancement of the polyol pathway, may be more cru- cial for the reductions of DRG neuronal attachment to the ECM proteins in diabetic mice. It is recognized that the increase in AR activity competes with nitric oxide (NO) synthase or glutathione reductase for NADPH [7,16,17]. Since NO is a potent vasodilator, the inhibition of NO synthase by the activation of AR and subsequent decrease in NO in nervous tissue can cause diminished nerve blood flow and nerve ischemia [16,17]. Reduced glutathione (GSH) is also important for cells to withstand oxidative stress, and a decrease in GSH by glutathione reductase inhi- bition may augment superoxide and hydrogen peroxide pro- duction in nervous tissue [7]. Consequently, a series of these changes due to the activation of AR at an early stage of diabetes could affect the structure and function of neuronal cell-surface receptors. Based on the present findings, the impaired binding of cell-surface receptors to the ligands of ECM proteins could be considered to be one of the initial functional disorders of peripheral sensory neurons in dia- betes. Thus, we speculate that ONO-2235 can prevent and/ or ameliorate diabetic sensory neuropathy, if it is adminis- tered to patients with diabetes of short duration.
In conclusion, administration of the aldose reductase inhi- bitor ONO-2235 to mice affected with STZ-diabetes for a short time restored the reductions of DRG neuronal attach- ment to the ECM proteins in vitro. These results suggest that initial metabolic disorders due to the activation of AR in diabetic mice may detrimentally alter the cell-surface recep- tors of DRG neurons for the ECM proteins.

We thank Dr. Jane Clarkin for reading the manuscript and Ono Pharmaceutical Company for the gift of ONO- 2235. This work was carried out with the financial support from a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and from Funds for Comprehensive Research on Aging and Health, Japan Health Science Foundation.

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