Exogenous neuritin treatment improves survivability and functions of Schwann cells with improved outgrowth of neurons in rat diabetic neuropathy

Abstract Pathogenesis and treatment for diabetic neuropathy are still complex. A deficit of neurotrophic factors affecting Schwann cells is a very important cause of diabetic neuropathy. Neuritin is a newly discovered potential neurotrophic factor. In this study, we explored the effect of exogenous neuritin on survivability and functions of diabetic Schwann cells of rats with experimental diabetic neuropathy. Diabetic neuropathy was induced in rats. 12‐week diabetic rats contrasted with non‐diabetic normal rats had decreased levels of serum neuritin and slowed nerve conduction velocities (NCVs). Schwann cells isolated from these diabetic rats and cultured in high glucose showed reduced cell neuritin mRNA and protein and supernatant neuritin protein, increased apoptosis rates, increased caspase‐3 activities and progressively reduced viability. In contrast, exogenous neuritin treatment reduced apoptosis and improved viability, with elevated Bcl‐2 levels (not Bax) and decreased caspase‐3 activities. Co‐cultured with diabetic Schwann cells pre‐treated with exogenous neuritin in high glucose media, and diabetic DRG neurons showed lessened decreased neurite outgrowth and supernatant NGF concentration occurring in co‐culture of diabetic cells. Exogenous neuritin treatment ameliorated survivability and functions of diabetic Schwann cells of rats with diabetic neuropathy. Our study may provide a new mechanism and potential treatment for diabetic neuropathy.

addition, a deficit of neurotrophic factors affecting Schwann cells (SCs) is another important cause of diabetic neuropathy. [8][9][10] Demyelination of fibres constitutes one of the main pathologic characteristics of diabetic neuropathy 6,11. SCs are essential to maintain normal structures and functions of the peripheral nervous system: formation of myelin around axons, the conduction of nervous impulses along axons, and nerve development and regeneration.. 12,13 Therefore, reduced viability of SCs may significantly contribute to peripheral nerve deficits in certain conditions. Viability of SCs is compromised, leading to demyelination, as nerve fibres are exposed to unfavourable conditions including hyperglycaemia. 11 Hyperglycaemia also results in apoptosis of SCs in vitro, 14,15 and severe hyperglycaemia-induced apoptosis can be ameliorated by a treatment of an exogenous neurotrophic factor. 16 So far, several neurotrophins have been found to be underproduced and involved in survival and functions of SCs in a diabetic or hyperglycaemic state. [17][18][19] Apart from these, our recent findings indicate that neuritin may be a potential novel neurotrophic factor affecting SCs' viability. 20 Neuritin, a small and highly conserved GPI-anchored protein, is present in the central nervous system, involving neurite outgrowth and maintenance of central nervous system development. 21,22 It is also expressed in dorsal root ganglia (DRG), involving axonal regeneration, and it is deficient in DRG neurons and axons in experimental diabetic neuropathy. 23 However, neuritin in relation to SCs in diabetic neuropathy has not been investigated. Recently, we discovered that neuritin existing in insoluble and mainly soluble (secreted) forms was expressed in SCs, and it was down-regulated and associated with apoptosis of SCs exposed to very high glucose milieu in vitro, which were prevented by exogenous IGF-1 (insulin-like growth factor-1) treatment. 20,24 In the present study, we established diabetic neuropathy model in rats as previously described 23,25 and cultured SCs and neurons isolated from the rats with and without diabetic neuropathy to explore, for the first time, the relation of compromised survival of SCs with down-expressed neuritin to experimental diabetic neuropathy and the effect of exogenous neuritin treatment on survivability and functions of the diabetic SCs.
Our study may provide a new mechanism of diabetic neuropathy and a new intervention with neuritin for diabetic peripheral neuropathy.

| Animals
All animals were obtained from Nanjing University Laboratory Animal Center. All experiments were conducted in accordance with Nanjing Medical University Regulations and with the Animals ACT.

| Diabetes induction
Adult male Sprague Dawley rats, ageing 7 ± 1 w, weighing 210 ± 10 g, were used for the study. At the beginning of the experiment, they were housed at 22 ± 2°C with 12-h alternating light/dark cycles and fed with standard laboratory rat chow. After an overnight fast, diabetes was induced in rats by a single intraperitoneal injection of STZ (Sigma, St. Louis, MO, USA) freshly dissolved in normal saline, at a dose of 55 mg/kg of bodyweight. Rats that received normal saline alone by the same route served as the control. Blood glucose was monitored from tail vein blood (OneTouch glucometer, Johnson & Johnson, New Brunswick, New Jersey, USA). STZ-treated rats with fasting blood glucose > 16 mmol/L were accepted for the study. Rats were randomly divided into normal or diabetic groups and group-housed with full access to food and water for 12 weeks. HbA1c (haemoglobin A1c), reflecting the mean glucose level over the past 12 weeks, was measured using high-performance liquid chromatography (Bio-Rad D10).Values of HbA1c were expressed as both % and mmol/mol.

| Electrophysiological study
Each rat was anaesthetized with a peritoneal injection of pentobarbital sodium (30 -40 mg/kg). Electrophysiological studies were performed using EMG (Viking IV, Nicolet) as previously described. 19 Briefly, the body temperature was maintained at 30°C with a heating lamp and controlled by a contact thermometer. The sciatic nerve was stimulated, and motor nerve conduction velocity (MNCV, m/s) was recorded from the first interosseous muscle of the hind paw.
Sensory nerve conduction velocity (SNCV, m/s) was recorded in the digital nerve to the second toe.

| Enzyme-linked immunosorbent assay
Serum samples of rats or supernatant samples of SC culture were collected and processed according to assay kit manufacturer's instructions.
Briefly, samples were centrifugated and added to microplates with wells coated with anti-neuritin (Boston Biochem) or anti-NGF (Thermo Fisher Scientific) where they were incubated at 37°C for 30 minutes.
Horseradish peroxidase (HRP) conjugate and tetramethylbenzidine reagents were added to the plates. Absorbance (OD) was read at 450 nm on the same microplate reader (Bio-Rad). Neuritin concentrations were measured and expressed as ng/ml in each sample, respectively. Culturesupernatant neuritin (secreted) or NGF concentrations were expressed as ng/g of total cellular protein using the Bradford method.

| SC culture and treatment
Schwann cells were isolated and purified from the sciatic nerves of these normal control or diabetic rats as previously described. 20,24 Briefly, after whole epineurium was stripped off, epineurium-free nerve tissue was collected and transferred to petri dishes where the dissociation solution was added (DMEM, 10% FCS, 1% pen/ strep, 0.125% collagenase, and 1.25 U mL/1 dispase) and incubated for 20 hours at 37°C, and the dissociated tissues were transferred and separated into single cells that were centrifuged, resuspended and seeded. Cells were initially plated on 6-cm petri dishes coated progesterone (20 nmol/L). To this serum-free medium, glucose was added: 5.6 mmol/L glucose mimicking normal glucose condition and 25 mmol/L glucose mimicking high glucose condition, respectively. Exogenous recombinant neuritin (5 or 10 ng/mL) (PeproTech) was used to treat SCs in certain conditions. For each experiment, SCs grown in growth media were first washed with PBS (Gibco) and cultured in conditioned serum-free media for certain length of time.
Experimental SCs were basically grouped into: (a) NSC, normal control SCs, isolated from normal rats and cultured in normal glucose

| Q-PCR measurement
Q-PCR measurement was conducted as previously described. 20,24 Briefly, following reverse transcription to cDNA, the PCR reac-

| Western blotting
Western blotting was conducted largely as previously reported. 20,24 Briefly, SCs were collected and homogenized in ice-cold lysis buffer.

| Annexin V/PI apoptosis assay
As previously described, 20,24 Apoptosis Assay Kit (Invitrogen) for flow cytometry was used to analyse apoptosis. Briefly, following TA B L E 1 BG, HbA1c, BW, serum neuritin and NCVs in rats at week 12

| TUNEL apoptosis assay
Fluorescein isothiocyanate-labelled TUNEL assay (Roche) was conducted as previously described. 20,24 Briefly, firstly, SCs on chamber slides were fixed with 4% paraformaldehyde, and incubated with fluorescein isothiocyanate-labelled TUNEL, at 37°C for 1 hour in the dark. The slides were counterstained with propidium iodide for 15 minutes on ice and treated with terminal deoxynucleotidyl transferase or without (but with the same volume of label solution), respectively. Positive controls were treated with DNase I before labelling. Samples were studied using ImageXpress Velos Laser Scanning Cytometer (Molecular Devices). Data were expressed as the percentage of TUNEL-positive cells relative to the total cell population in each group.

| Co-culture of SCs and neurons
Isolated SCs were cultured as above and seeded at a density of 2 × 10 5 /mL into 35-mm petri dishes with the growth media.

| Immunocytochemistry and neurite outgrowth analysis
Co-cultured cells were fixed with ice-cold 4% paraformalde-

| Statistical analysis
All above measurements were repeated 3 times in each independent experiment. Data were expressed as mean ± SE of 3-6 independent experiments. One-way ANOVA for multiple comparisons of quantitative mRNA and proteins, and post hoc test and chi-square test for percentage comparisons of apoptosis rates were conducted. P value < .05 was considered to be significant.

| Increased BG, decreased serum neuritin and slowed NCVs in diabetic rats
Diabetic rats showed polydipsia, polyuria, polyphagia, muscle wasting and progressive loss of bodyweight over 12 weeks of diabetic course. During this period, diabetic rats had an obviously elevated F I G U R E 3 Apoptosis in Schwann cells. Isolated from sciatic nerves of diabetic rats with decreased serum neuritin and slowed NCVs, Schwann cells in culture for 48 h showed increased apoptosis rates using Annexin V/PI assay (A and B) and TUNNEL assay (C) in contrast to those from normal control rats, respectively. These diabetic Schwann cells in culture treated with exogenous neuritin had reduced apoptosis rates. The apoptosis rates decreased with an increasing dose of neuritin treatment (from 5 to 10 ng/mL) (A, B, and C). NSC, normal control Schwann cells, isolated from normal rats and cultured in normal glucose (5.6 mmol/L). DSC, diabetic Schwann cells, isolated from diabetic rats and cultured in high glucose (25 mmol/L). DSC + NEU, DSC treated with exogenous neuritin (5 or 10 ng/mL). A: The examples of apoptotic Schwann cells using Annexin V/PI assay and phase-contrast pictures of Schwann cells (20 × magnification), with the right lower quadrant showing early apoptotic cells and the left lower quadrant showing viable cells in each graph, respectively. B and C: DSC vs NC, *P < .01; DSC + NEU vs DSC, **P < .05, ***P < .01, respectively. No statistical differences were found between NSC and DSC + NEU 10 ng/ mL. Data were expressed as mean ± SE of 6 independent experiments level of blood glucose that was monitored stable and HbA1c that reflected mean blood glucose concentrations over the past 12 weeks.
In these diabetic rats, increasingly decreased levels of serum neuritin and gradually slowed velocities of both motor and sensory nerve conduction were observed, which started at week 2 of the experiment (data not shown). These changes in diabetic rats were contrasted with non-diabetic normal rats, in particular at week 12 of diabetic course (Table 1).

| Decreased neuritin expression, increased apoptosis and decreased viability of diabetic SCs
Neuritin was localized using immunostaining in the cytoplasms of  Figure 2C), compared to SCs isolated from normal agecontrolled rats and cultured in normal glucose. In a small percentage (nearly 14%) of these diabetic SCs, early apoptotic changes were found using Annexin V/PI and TUNEL assays, and this was obvious if compared to non-diabetic normal cells cultured in a normal glucose condition ( Figure 3A,C). In addition, a dynamic assessment of the viability (%) of SCs using CCK8, reflecting the number of viable cells and the ability of cell proliferation, showed that diabetic SCs had lower and progressively lower viability, from nearly 80% to 60%, compared to normal SCs over 4 days from day 1 to day 4 in respective cultures ( Figure 4A).

F I G U R E 4 Viability of Schwann cells. Isolated from sciatic nerves of diabetic rats with decreased serum neuritin and slowed
NCVs, Schwann cells showed progressively decreased relative viability (%) over 4 days in culture in contrast to those from normal control rats (A). These diabetic Schwann cells in culture treated with exogenous neuritin had reduced compromised viability using CCK-8. The viability increased with an increasing dose of neuritin treatment (from 5 to 10 ng/mL) (B). NSC, normal control Schwann cells, isolated from normal rats and cultured in normal glucose (5.6 mmol/L). DSC, diabetic Schwann cells, isolated from diabetic rats and cultured in high glucose (25 mmol/L). DSC + NEU, DSC treated with exogenous neuritin (5 or 10 ng/mL). A, DSC vs NSC, *P < .05, **P < .005, respectively. B, DSC + NEU (5 ng/mL) vs DSC, *P < .05; DSC + NEU (10 ng/mL) vs DSC, **P < .01, ***P < .005, respectively. No statistical differences were found between NSC and DSC + NEU 10 ng/mL. Data were expressed as mean ± SE of 6 independent experiments F I G U R E 5 Caspase-3 activity of Schwann cells. Isolated from sciatic nerves of diabetic rats with decreased serum neuritin and slowed NCVs, Schwann cells in culture for 48 h showed an increased relative activity (folds) of caspase-3 in contrast to those from normal control rats. These diabetic Schwann cells in culture treated with exogenous neuritin had a decreased activity of caspase-3 compared to without. Caspase-3 activity decreased with an increasing dose of neuritin treatment (from 5 to 10 ng/mL). NSC, normal control Schwann cells, isolated from normal rats and cultured in normal glucose (5.6 mmol/L). DSC, diabetic Schwann cells, isolated from diabetic rats and cultured in high glucose (25 mmol/L). DSC + NEU, DSC treated with exogenous neuritin (5 or 10 ng/mL). *: DSC vs NSC, P < .01. **: DSC + NEU vs DSC, P < .05. ***: DSC + NEU vs DSC, P < .01. No statistical differences were found between NSC and DSC + NEU 10 ng/mL. Data were expressed as mean ± SE of 6 independent experiments

| Improved apoptosis and viability of diabetic SCs treated with exogenous neuritin
Schwann cells isolated from these 12-week diabetic rats with serum neuritin concentrations and slowed nerve conduction velocities were treated with exogenous neuritin for 48 hours in high glucose culture. These neuritin-treated diabetic SCs showed decreased apoptosis rates by nearly 25%, compared to untreated diabetic SCs cultured in high glucose condition ( Figure 3B,C).
Consistently, the viability (%) of these neuritin-treated diabetic SCs was shown gradually increased from 20% to 40%, compared to untreated SCs over 4 days from day 1 to day 4 in respective cultures, although the viability was not obviously different between them at day 1 and the obvious difference started to be shown from day 2 ( Figure 4B). In addition, apoptosis and viability of SCs were increasingly improved with an incrementing dose of exogenous neuritin treatment. In general, the extent of the effect of 10 ng/mL exogenous neuritin was greater than 5 ng/mL exogenous neuritin ( Figures 3B,C, and 4B).

| Depressed caspase-3 and elevated Bcl-2 in diabetic SCs treated with exogenous neuritin
These diabetic SCs that were treated with exogenous neuritin for 48 hours in high glucose culture showed decreased activities of caspase-3 and elevated levels of Bcl-2 protein compared to untreated diabetic SCs (Figures 5 and 6). Like its effects on apoptosis and viability of SCs, exogenous neuritin treatment depressed the activity of caspase-3 and increased the level of Bcl-2 (not Bax) in a dosedependent manner. In contrast, untreated diabetic SCs showed an increased activity of caspase-3 and a decreased level of Bcl-2 protein (not Bax) compared to non-diabetic normal SCs.

| Improved neurite outgrowth of neurons in co-culture with diabetic SCs pre-treated with exogenous neuritin
Dorsal root ganglia neurons, which were isolated from diabetic rats with decreased serum neuritin and slowed NCVs and co-cultured with diabetic SCs for 48 hours in high glucose media, showed a reduction of both longest neurite outgrowth ( Figure 7A,B) and average length of neurite outgrowth ( Figure 7C), and supernatant NGF concentration ( Figure 7D), compared to those DRG neurons co-cultured with SCs from normal control rats in normal glucose media.
In contrast, these diabetic DRG neurons co-cultured with diabetic SCs that were pre-treated for 48 hours with exogenous neuritin before co-culture had less reduction of two measurements of neurite outgrowth ( Figure 7B,C) and supernatant NGF concentration ( Figure 7D). These parameters increased with an increasing dose of neuritin treatment (from 5 to 10 ng/mL), although 5 ng/mL of exogenous neuritin treatment did not show a significant difference from without.

| D ISCUSS I ON
In this study, we demonstrated decreased serum neuritin concentrations in established neuropathy with obviously slowed nerve conduction velocities in rats with 12-week diabetic duration. Our previous study found neuritin was produced from SCs and pre- These diabetic Schwann cells in culture treated with exogenous neuritin had less reduction of Bcl-2 compared to without. Bcl-2 increased with an increasing dose of neuritin treatment (from 5 to 10 ng/mL). Bax did not have these changes in respective cultures (A and B). NSC, normal control Schwann cells, isolated from normal rats and cultured in normal glucose (5.6 mmol/L). DSC, diabetic Schwann cells, isolated from diabetic rats and cultured in high glucose (25 mmol/L); DSC + NEU, DSC treated with exogenous neuritin (5 or 10 ng/mL). A, The example of Bcl-2, Bax and β-actin bands using Western blotting. B, DSC vs NSC, *P < .01; DSC + NEU vs DSC, **P < .05, ***P < .01, respectively. No statistical differences were found between NSC and DSC + NEU 10 ng/mL. Data were expressed as mean ± SE of 6 independent experiments rats with diabetic neuropathy. More importantly, the decreased neuritin production is associated with reduced survival of SCs. In one separate study (unpublished), we used lentivirus transfection to silence neuritin gene of SCs isolated from normal rats, leading to nearly 70% drop of neuritin production with nearly 60% rise of apoptosis rate. 20 The present study showed that diabetic SCs  was not explored in this study. We did not find significant osmotic effects of high glucose on these SCs (mannitol control, data not shown). Insulin-like growth factor-1 (IGF-1, not measured in this study) maybe one mediator since we observed that IGF-1 levels reduced in experimental diabetes 19,27 and, in vitro, exogenous IGF-1 stimulated neuritin expression in SCs. 24 It is unclear what the relationship is between neuritin and other hyperglycaemia-induced factors leading to diabetic neuropathy: a deficiency of other neurotrophins, [17][18][19] enhanced polyol pathway activity, 5,6 increased non-enzymatic glycation 7 and augmented oxidative stress among others.
From regenerative or rehabilitative perspective, a deficit of neurotrophic factors affecting SCs is a very important cause of diabetic neuropathy. In this respect, our study provided evidence of a novel neurotrophic factor-neuritin deficit in the development of experimental diabetic neuropathy. Neuritin replenishment may be applied to practical in vivo treatment for diabetic neuropathy, if metabolism of neuritin is fully understood.

ACK N OWLED G EM ENTS
This project was supported by National Natural Science Foundation

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data were available and presented in the main manuscript.