Plasminogen repairs abnormal pain perception through improving sensory function recovery and regeneration of peripheral small nerve fiber in db/db mice

Painful diabetic peripheral neuropathy (PDPN) is a devastating complication of diabetes and severely threatens the health of humankind. The plasminogen activator system and plasminogen (Plg) have multiple functional roles in tissue regeneration and extracellular matrix remodeling, which suggests that Plg may have a potentially pivotal role in anti-PDPN. In the present study, we explore whether an increased level of circulating Plg has positive effect on repairing abnormal pain perception in diabetic mice model. Our data demonstrated that additional Plg not only helps healing pain allodynia or hyperalgesia on the mice at the age of 8 weeks old in early PDPN, but more important, also has positive effects of regaining normal pain perception from hypoalgesia on the mice at ages of 14-15 or 24-25 weeks in advanced PDPN. Furthermore, our data also reveal a possible mechanism for Plg’s contribution to rebuilding normal pain perception among db/db mice by promoting axonal myelination and regeneration of small nerve fiber in peripheral nervous system. Therefore, our data suggest that Plg show promise to become a drug candidate for treating diabetic peripheral neuropathic pain.

grouped and treated with Plg for the experiments. If not mentioned, at least five mice were included in each experimental group.

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h-Plg protein administration in diabetic burn-wound healing 103 and diabetic peripheral neuropathy study 104 For the study of diabetic burn-wound healing, the male mice at age 16-26 weeks old 105 were anesthetized by an intraperitoneal injection of 50 mg/Kg sodium pentobarbital. A 106 copper rod was heated to 95-100°C by submersion in boiling water. The copper rod was 107 immediately applied vertically for 6 seconds without additional pressure on the back skin 108 of mice that had also been depilated before wounding. After wounding, all mice were 109 individually caged, and wounds were neither sutured nor dressed. The mice received 110 standardized wounds, and then 2 mg of human Plg was administered daily by 0.2ml IV 111 injection. In the control group, 0.2 ml of PBS was administered daily by IV injection as a 112 placebo. The daily treatments were continued for indicated days depending on the 113 experimental design.

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h-Plg protein administration in diabetic peripheral neuropathy 115 study 116 As for the study of diabetic peripheral neuropathy, each group of male mice at the age examined: allodynia, hyperalgesia, and hypoalgesia in response to cold and mechanical 125 stimuli. To quantify mechanical sensitivity of the foot, the standard quantitative sensory 126 testing (QST) was used to record numbers of brisk foot withdrawal in response to 127 normally noxious and innocuous mechanical stimuli as described previously [16][17].

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To quantify cold sensitivity of the foot, brisk foot withdrawal in response to acetone to a syringe. The bubble was then gently touched to the heel. The acetone quickly spread expressed as a percent: (# of trials accompanied by brisk foot withdrawal) X 100 / (# of 149 total trials). Cold sensitivity was tested on each mouse on day 0 (1 day before 150 administration of Plg) and 3, 4, 6, 7, 11, 12, and 16 days after treating with Plg.

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To quantify the pain sensation evoked by pin-prick, the 27-gauge needle was used to

H&E staining
160 The sciatic nerve tissues were fixed in 4% paraformaldehyde, embedded in paraffin 161 and sectioned 3 µm thick. The sections were stained for morphological analysis using an 162 H&E staining kit. The slides were examined by light microscopy under a Nikon 163 microscope, and images were recorded digitally using a camera connected to a computer.

Immunohistochemical analyses
165 The paraffin-embedded sections were rehydrated and then treated with antibodies peroxidase anti-peroxidase method. In brief, the antigens were first retrieved by treatment 50) and incubated with the antibodies against Fibrin or PGP9.5 diluted in PBS. After this 172 procedure, an anti-rabbit link antibody was applied, followed by a rabbit PAP complex.

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The staining was visualized through a diaminobenzidine (DAB) reaction, and the sections 174 were counterstained with hematoxylin.

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Light microscopic examination 176 The slides were examined by light microscopy under a Nikon microscope (C-SHG1),

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and images were recorded digitally using Nikon DS-Fi3 connected to a computer. When skin is increased after administration with Plg for 14 days (Fig.1). This results indicated 289 that Plg promoted small nerve fibers regeneration at the wounded site in the diabetic mice.

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Blasi and Mignatti have disclosed that the PA system was implicated in various tissue 291 remodelling processes as early as 1990s' [28][29]. That is to say Plg can improve 292 allodynia and hyperalgesia of early stage of PDN through promoting the regeneration of 293 small nerve fibers to elevate the pain perception threshold.

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As the disease course progresses, mild segmental axonal demyelination then occurs, 295 followed by frank axonal degeneration of myelinated fibers as demyelination surpasses  (Fig. 2B). In addition, the deposition of fibrin in sciatic nerve tissue of the Plg-treated 313 group was significantly decrease (Fig.2C)