Demonstration of therapeutic effect of plasma-synthesized polypyrrole/iodine biopolymer in rhesus monkey with complete spinal cord section

Abstract

Spinal cord injury (SCI) can cause paralysis, and although multiple therapeutic proposals have been developed in murine models, results have hardly been replicated in humans. As non-human primates (NHP) are more similar to humans than rodents, the current study investigated whether it was possible to reproduce in a NHP, the previously obtained beneficial results by using a plasma-synthesized polypyrrole/iodine (PPy/I) biopolymer, which reduce glial scar formation and inflammatory response and promotes nerve tissue preservation, regenerative processes and functional recovery in rats. In NHPs (Rhesus monkey) with SCI by complete transection (SCT) and with plasma-synthesized PPy/I application (experimental) or without (control), the expression of pro-inflammatory cytokines in blood, preservation of nervous tissue through magnetic resonance imaging and histological and morphometric techniques, regeneration through immunohistochemistry study and functional recovery through clinical examination, were evaluated. Control NHP showed a markedly increased of pro-inflammatory cytokines vs. experimental NHP, which preserved more nerve tissue. At the end of the follow-up, a thinner glial scar in the injured spinal cord was observed in the experimental NHP as well as regenerative nerve processes (NeuN and β-III tubulin expression), while control NHP had a marked glial scar, large cysts and less nerve tissue at the injured zone. Plasma-synthesized PPy/I also reduced the loss of pelvic limb muscle mass and allowed the experimental NHP recovered knee-jerk, withdrawal and plantar reflexes as well as movement in the hind limbs. Since most of the beneficial effects of plasma-synthesized PPy/I previously reported in rats were also observed in the NHP, these preliminary findings make their replication in humans with SCI more likely.

Fig. 1

Characterization of plasma-synthesized polypyrrole-Iodine (PPy/I) biopolymer. A Fourier Transform Infrared (FT-IR) transmittance spectrum; B Scanning electron microscopy (SEM) photomicrograph of PPy/I particles obtained after grinding a thin film; C Elemental atomic % of carbon, oxygen and nitrogen in PPy/I

Fig. 2

Evaluation of pro-inflammatory cytokines IL-2 (A), IL-4 (B), IL6 (C), GM-CSF (D), IFɣ (E) and TNFα (F) in plasma after a complete spinal cord transection without plasma-synthesized polypyrrole/iodine (PPy) application (SCT) or with PPy application (SCT/PPy) in nonhuman primates (NHPs). The analysis was performed before (basal) and 24 h, 72 h, and 12 weeks after SCT. All evaluated cytokines increased in the NHP without PPy application, while they decreased or maintain their basal levels in the experimental NPH with PPy application

Fig. 3

T2-weighted magnetic resonance imaging study shows the whole spinal cord. A, F Before spinal cord transection (SCT) in non-human primates (NHPs), immediately after SCT, and 4, 8, and 12 weeks after SCT without (B–E) or with (G–J) application of plasma-synthesized polypyrrole/iodine (PPy). The images of the control NHP show loss of nerve tissue continuity and clear separation of the proximal and distal ends of the injured spinal cord, while the image of the experimental NHP with PPy application, do not show this same loss of continuity since the plasma-synthesized PPy biopolymer was identified between the two stumps of the spinal cord shortening or closing the space generated between them after SCT

Fig. 4

Morphometric analysis of semi-automatic spinal cord segmentation using magnetic resonance imaging. On the top, images of semiautomatic segmentation of the spinal cord, where the boundaries of gray matter, white matter, cerebrospinal fluid, scar, cysts, and plasma-synthesized polypyrrole/Iodine (PPy) were defined by regions of interest (ROIs) in the non human primates (NHP) before spinal cord injury (A and F) and at different times after injury in the untreated NHP (SCI) (B–E) and in the NHP treated with PPy/I (SCI+PPy) (F–J). The volumes were calculated by means of the polygonal approximation method, the surfaces were rendered and results were expressed in mm³. In the middle part, morphometric analysis of the images showing the differences between the experimental NHP (with PPy application) and the control NHP (without PPy application) in relation to the percentage of spinal cord tissue (spine), other tissue (OT), plasma-synthesized polypyrrole/Iodine implant (PPy), glial scar (scar) and cysts (cysts) in the injured spinal cord. On the bottom, graphical representation of morphometric analysis is showed in percentage

Fig. 5

Photomicrographs of sagittal sections from transected spinal cord of control non-human primate (NHP) without plasma-synthesized polypyrrole/iodine (PPy) application after spinal cord transection (SCT) and experimental NHP with PPy application (SCT/PPy). Spinal cord cephalic zone (A–C and G–I) and spinal cord caudal zone (D–F and J–L) show changes in nervous tissue cytoarchitecture of NHPs: presence of inflammatory cells including polymorphonuclear and macrophages (arrows), microcysts, cysts and cavitations (stars) and glial scar (arrowheads). PPy relation with the spinal cord tissue in SCT/PPy is shown (triangle). Hematoxylin-eosin staining. Scales bars = 1 mm, 100 µm and 50 µm, respectively

Fig. 6

Photomicrographs of sagittal sections from spinal cord of control non-human primate (NHP) without plasma-synthesized polypyrrole/iodine (PPy) application after spinal cord transection (SCT) and of experimental NHP with PPy application (SCT/PPy). Spinal cord cephalic zone (A–C and G–I) and spinal cord caudal zone (D–F and J–L) show presence of collagen fibers on stumps of injured spinal cord and meninges around the epicenter of injury in bluish green color (arrows), and microcysts, cysts, and cavitations (stars). PPy relation with the spinal cord tissue in SCT/PPy is shown (triangle). Masson trichrome staining. Scales bars = 1 mm, 100 µm and 50 µm, respectively

Fig. 7

Photomicrographs of sagittal sections from transected spinal cord of control non-human primate (NHP) without plasma-synthesized PPy application after spinal cord transection (SCT) and of experimental NHP with PPy application (SCT/PPy). Spinal cord cephalic zone (A–C and G–I) shows a better myelination of nerve fibers (blue color) than in caudal zone (D–F and J–L) and more myelinated fibers in experimental NHP than in control. Microcysts, cysts, and cavitations are larger in the control NHP. Polypyrrole/iodine (PPy) is shown in brown. Luxol fast blue staining. Scales bars = 1 mm, 100 µm and 50 µm, respectively

Fig. 8

Photomicrographs of sagittal sections from transected spinal cord of control non-human primate (NHP) without plasma-synthesized PPy application after spinal cord transection (SCT) and of experimental NHP with PPy application (SCT/PPy). Spinal cord cephalic zone (A–C and G–I) shows myelinated fibers (magenta color) crossing caudal zone (D–F and J–L) where more myelinated fibers in experimental NHP than in control NHP were seen, as well as oligodendrocytes cells (red color) close to the myelinated axons, glial fibers (light green), astrocytes (green), nucleus (dark blue), collagen (blue) and neurons (dark blue). Microcysts, cysts, and cavitations (asterisks) and Polypyrrole/iodine (PPy) relation with the spinal cord tissue (triangle). Lapham staining. Scales bars = 1 mm, 100 µm and 50 µm, respectively

Fig. 9

Photomicrographs of sagittal sections from spinal cord cephalic (A–C) and caudal (D–F) zones from control non-human primate (NHP) without plasma-synthesized polypyrrole/iodine (PPy) application after spinal cord transection (SCT) and of experimental NHP with PPy application (SCT/PPy). Spinal cord cephalic (G–I) and caudal (J–L) zones of spinal cord from experimental NHP with PPy application show the presence of neurons on nerve tissue and around of the PPy application zone (arrows). PPy relation to the spinal cord tissue can be observed as well as microcysts, cysts, and cavitations. Golgi argentic impregnation technique. Scales bars = 1 mm, 100 µm and 50 µm, respectively

Fig. 10

Expression of βIII-tubulin and NeuN in spinal cord sagittal sections from transected spinal cord of control non-human primate (NHP) without plasma-synthesized polypyrrole/iodine (PPy) application (SCT) and experimental NHP with PPy/I application (PPy). A Photomicrographs of βIII-tubulin (red channel) and NeuN (green channel) immunofluorescence counterstained with DAPI (blue channel) in control and experimental NHPs. B Quantification of number of NeuN+ cells. C Quantification of βIII-tubulin expression area (red channel). B, C were quantified in 10 sections of 1 mm length considering the epicenter of the lesion plus 5 mm in cephalic direction and 5 mm in caudal direction. Scale bar = 1 mm in panoramic micrographs and 200 µm in detailed micrographs