Figure 1.
Flow-chart of prevention and therapeutic protocols in streptozotocin-induced diabetic neuropathy.
Diabetes was induced by intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin (STZ). Bevacizumab (10 or 20 mg/kg) was administered i.p. at the indicated times. Four groups of Sprague Dawley rats (8 per group) were used in both the protocols (untreated, STZ untreated, STZ+bevacizumab 10 mg/kg, STZ+bevacizumab 20 mg/kg). In the prevention protocol, bevacizumab was administered at week 1 (after confirmation of diabetes), and week 4; rats were sacrificed at week 8. In the therapeutic protocol, bevacizumab was administered at week 8 and 12, and rats were sacrificed at week 16. Nerve conduction velocity (NCV) and nociceptive threshold assessment were measured at the indicated times.
Figure 2.
Hyperglycaemia did not increase DRG neuron or Schwann cell (SC) apoptosis.
(A) Neuron-SC co-cultures showed a modest increase of apoptosis rate only at the highest glucose concentration; paclitaxel (tax) and cisplatin (cis) exposition was used as positive controls. (B) In DRG neuron monocultures, hyperglycaemia did not induce apoptosis. (C) Tubulin-III and GFAP staining demonstrated that apoptosis mainly involved SC. (D) SC monocultures showed a mild increase of apoptosis rate at the highest glucose concentrations, similar to that observed in co-cultures (see C). (E) Flow cytometry by annexin V/PI assay confirmed the absence of apoptosis in co-cultures exposed to hyperglycaemia. (F) Representative cytogram showing the absence of apoptosis in control co-culture (ctrl) and after 24 hour exposition to hyperglycaemia 45 mM (hg), compared to the high apoptosis rate after exposition to anti-neoplastic compounds (tax, cis). Data are expressed as mean±SEM of independent experiments (n = 8) *p<0.05; **p<0.005; ***p<0.0005 vs controls; #p<0.05; ##p<0.005; ###p<0.0005 vs SC controls.
Figure 3.
Hyperglycaemia did not affect mitochondrial membrane potential.
(A) JC-1 fluorescence emission measurements showed a reduction in red-to-green ratio in neurons exposed to cisplatin (cis) but not to hyperglycaemia (45 mM) nor paclitaxel (tax) (n = 4), *p<0.05 vs ctrl. (B) Representative flow cytogram of mitochondrial membrane potential in co-cultures using JC-1. The shift of JC-1 fluorescence from red (FL2) to green (FL1) indicates a collapse of mitochondrial membrane potential.
Figure 4.
Hyperglycaemia significantly and in a dose-dependent fashion affected neurite outgrowth.
(A) Exposure to increasing glucose concentrations induced a significant dose-dependent decrease of neurite outgrowth in neuron-SC co-cultures (B) but not in neuron monocultures. (C) Exposure of neuron monocultures to hyperglycaemia-conditioned SC monoculture medium caused a significant decrease of the neurite outgrowth. All data are normalized to control and presented as mean±SEM of independent experiments (n = 8) *p<0.05; **p<0.005; ***p<0.0005 vs control. (D) Cytokine array of hyperglycaemia-conditioned SC monoculture medium showing increase of VEGF compared to control. (E) Data are expressed as relative levels of VEGF.
Figure 5.
Hyperglycaemia-induced post-translational regulation of VEGF.
(A) Exposure to glucose 45 mM for 24 hours increased the level of secreted VEGF in neuron-SC co-cultures and SC monocultures. Data are express as percentage difference between their own control in independent experiments (n = 5) *p<0.05. (B) Hyperglycaemia significantly increased VEGF mRNA only in neuron monocultures (n = 5). (C) Representative and (D) quantitative western blot (WB) demonstrating VEGF decrease in hyperglycaemia-conditioned SC monocultures compared with control SC monocultures. VEGF level was not affected in neuron monocultures. Data are expressed as mean±SEM of independent experiments (n = 4) *p<0.05 vs controls. (E) VEGF induced a significant reduction in axonal outgrowth in neuron/Schwann cells coculture. Data are expressed as mean±SEM of independent experiments (n = 3) *p<0.05 vs controls.
Figure 6.
Bevacizumab prevented hyperglycaemia-mediated neurite outgrowth impairment and normalized FLT-1.
(A) bevacizumab prevented the decrease of hyperglycaemia-mediated neurite outgrowth in neuron-SC co-cultures (n = 5) and (B) the increase of FLT-1 mRNA in neurons monocultures (n = 5). (C) Representative and (D) quantitative western blot (WB) (n = 5) showing the significant decrease of FLT-1 in hyperglycaemia-conditioned SC monocultures and the preventive effect of bevacizumab. (E) sFLT-1 decreased in the medium of all neuron and SC monocultures and co-cultures exposed to hyperglycaemia (n = 5). (F) Bevacizumab did not affect sFLT-1 level in hyperglycaemia-conditioned co-cultures (n = 3). Data are expressed as mean±SEM of independent experiments. *p<0.05 vs controls; #p<0.05 vs hyperglycaemia; ##p<0.01 vs hyperglycaemia.
Figure 7.
Bevacizumab prevented and restored peripheral nerve functions in diabetic rats.
Control and STZ-diabetic rats were treated with bevacizumab according to the prevention (A, B, C) or therapeutic (D, E, F) schedule. (A) Bevacizumab prevented in a dose-dependent fashion thermal hypoalgesia, (B) mechanical threshold decrease and (C) nerve conduction velocity decrease in diabetic rats. In the therapeutic schedule, bevacizumab restored (D) thermal hypoalgesia (E) mechanical threshold and (F) nerve conduction velocity decrease in diabetic rats. Data are expressed as mean±SEM (n = 8 animals per group) *p<0.05 vs controls; **p<0.01 vs controls; ****p<0.001 vs controls; #*p<0.05 vs STZ; ##p<0.01 vs STZ.