Spinal cord regeneration therapy

Abstract

The disclosure relates to spinal cord regeneration therapy. In one aspect, the disclosure provides for administering to a subject suffering from spinal cord injury an effective amount of at least four herbal ingredients: polygala tenuifolia, astragalus membranaceus, ligusticum wallichii, and angelica sinensis, roots or rhizomes thereof, or extracts thereof. In other aspects, the disclosure provides in vitro and in vivo methods for determining the effectiveness of drugs and drug combinations and their effective amounts required to treat spinal cord injury.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 355,376, filed on June 24, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0002] The present disclosure relates to a method for promoting spinal cord regeneration, and more particularly, to inducing and / or accelerating recovery of spinal cord injury by administering to a subject an herbal composition comprising a combination of traditional Chinese herbal products or extracts thereof. Background Art

[0003] Spinal cord injury (SCI) is a serious medical problem. In severe cases, SCI may lead to loss of connection between the brain and spinal cord, resulting in paraplegia, quadriplegia or even death. SCI often causes devastating neurological deficits, which not only destroy motor, sensory and autonomic functions, but also lead to loss of bladder and bowel control, breathing problems, chronic pain and increased susceptibility to infection. This leads to poor quality of life and economic burden for SCI patients.

[0004] Although SCI is a leading cause of death and disability, there is a lack of effective and safe therapeutic agents to treat individuals with SCI. Therefore, new therapeutic approaches to treat SCI and related neurological deficits are urgently needed. More specifically, new therapies are needed to promote spinal cord regeneration and enable damaged or diseased nerves to function again. Summary of the invention

[0005] The following is provided to facilitate an understanding of some of the innovative features characteristic of the present disclosure. A full understanding of the various aspects of the present disclosure can be gained by considering the specification, claims, drawings, and abstract as a whole.

[0006] According to a first aspect, there is provided a method for treating a subject to induce and / or accelerate recovery from spinal cord injury, the method comprising administering to the subject an effective amount of a herbal composition comprising at least four herbal ingredients: Polygalae (thin leaf milkwort), Astragali (membranous milkvetch), Ligusticum Chuanxiong and Angelica sinensis (Chinese angelica), their roots or rhizomes or extracts thereof.

[0007] In one embodiment of the first aspect, the herbal composition of the method further comprises at least one additional herbal ingredient selected from the group consisting of Salviae Miltiorrhizae (red sage), Paeoniae Rubra (red peony), Carthamus Tinctorius flower (safflower), Semen Persicae (Prunus Persica seed) and Acori Tatarinowii (grassleaf sweet flag), their roots or rhizomes, or extracts thereof.

[0008] In one embodiment, the herbal composition of the method consists essentially of at least four herbal ingredients, their roots or rhizomes, or extracts thereof, and the four herbal ingredients are Polygala tenuifolia, Astragalus membranaceus, Ligusticum chuanxiong, and Angelica sinensis.

[0009] In one embodiment, the herbal composition of the method consists essentially of four herbal ingredients, their roots or rhizomes or extracts thereof, and the four herbal ingredients are Polygala tenuifolia, Astragalus membranaceus, Ligusticum chuanxiong and Angelica sinensis.

[0010] In one embodiment, the herbal composition of the method consists essentially of at least nine herbal ingredients, their roots or rhizomes or extracts thereof, and the nine herbal ingredients are Polygala tenuifolia, Astragalus membranaceus, Ligusticum chuanxiong, Angelica sinensis, Salvia miltiorrhiza, Paeonia lactiflora, Carthamus tinctorius flower (Carthamus tinctorius), Persica persica (Peach seed) and Acorus tatarinowii.

[0011] In one embodiment, the herbal composition of the method consists of a combination of extracts of nine herbal ingredients: Radix Polygalae, Radix Astragali, Rhizome of LigusticumChuanxiong, Angelica Sinensis, Radix et Rhizoma Salviae Miltiorrhizae, Paeonia Rubra, Carthamus tinctorius, Persica Semen, and Acorus tatarinowii.

[0012] In one embodiment, the herbal composition of the method is a pharmaceutical composition. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or excipients.

[0013] In a specific embodiment, the pharmaceutical composition used in the method herein comprises a combination of extracts of nine herbal ingredients, namely, Polygala tenuifolia, Astragalus membranaceus, Ligusticum chuanxiong, Angelica sinensis, Salvia miltiorrhiza, Paeonia lactiflora, Carthamus tinctorius, Semen persica and Acorus tatarinowii, and one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment, the pharmaceutical composition is MLC901 (also known as NeuroAiD II TM ).

[0014] In another specific embodiment, the pharmaceutical composition used in the method herein comprises a combination of extracts of four herbal ingredients, namely, Polygala tenuifolia, Astragalus membranaceus, Ligusticum chuanxiong and Angelica sinensis, and one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment, the pharmaceutical composition is MLC1501.

[0015] In one embodiment, recovery from spinal cord injury is induced and / or accelerated by neuronal cell regeneration.

[0016] In one embodiment, the herbal composition of the method promotes the growth of neuronal cells.

[0017] In one embodiment, the neuronal cell is a vertebrate neuronal cell.

[0018] In one embodiment, the vertebrate neuronal cells are human neuronal cells.

[0019] In one embodiment, the neuronal cells are derived from cortical neuronal cells.

[0020] In one embodiment, administration of the herbal composition results in regeneration of the spinal cord and / or results in regeneration of connections between the brain and the spinal cord and / or results in regeneration of damaged neuronal tissue or cells.

[0021] In one embodiment, administration of the herbal composition promotes recovery of limb movement in a paralyzed subject.

[0022] In one embodiment, an effective amount of the herbal composition is provided, the effective amount being about 1 mg / kg to about 100 mg / kg.

[0023] In one embodiment, a method is provided that includes administering a second agent for treating a subject having a spinal cord injury.

[0024] In one embodiment, the second agent is a drug effective for controlling pain and / or muscle spasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are included to more clearly illustrate specific embodiments of the present disclosure and related technologies. The accompanying drawings included herein provide a further understanding of the present disclosure and are incorporated in and constitute a part of the present disclosure. It should be understood that the accompanying drawings illustrate implementations of the present disclosure and are used together with the rest of the present disclosure to explain the principles of the present disclosure. Obviously, the accompanying drawings illustrate embodiments of the present disclosure, and a person of ordinary skill in the art can easily understand other embodiments from the accompanying drawings described herein.

[0026] Figure 1A flow chart is shown for the use of an in vitro spinal cord injury model in NSC-34 cell line culture, where injury is produced by mechanical ("In vitro Mechanical Injury") and chemical ("In vitro Chemical Injury") methods.

[0027] Figure 2 A schematic diagram of the in vitro mechanical injury method is shown.

[0028] Figures 3a and 3b show a flow chart for using an in vivo spinal cord injury model in adult SD rats, where the injury is produced by mechanical or chemical means.

[0029] Figure 4 Shown are the results of an in vitro mechanical injury method following treatment with different concentrations of MLC901: a) control cells with medium only, b) 800 μg / mL, c) 1000 μg / mL, d) 1200 μg / mL and e) 1400 μg / mL.

[0030] Figure 5 Shown is a bar graph of neurite outgrowth over 3 days following treatment with different MLC901 concentrations in an in vitro mechanical injury method.

[0031] Figure 6 Shown is a bar graph of mean neurite counts over 3 days following treatment with various MLC901 concentrations in an in vitro mechanical injury method.

[0032] Figure 7 Shown are time-lapse imaging over 3 days, reflecting how MLC901 promotes neurite outgrowth and neurite counts in the injured area in an in vitro mechanical injury approach.

[0033] Figure 8 Shown are time-lapse images reflecting neurite outgrowth at days 0 and 3 of NSC34 cells injured in an in vitro mechanical injury method.

[0034] Fig. 9 Shown is a bar graph of neurite outgrowth after time-lapse imaging at day 0, day 1, day 2, and day 3 in an in vitro mechanical injury method with different MLC901 concentrations.

[0035] Fig.10 Shown is NSC-34 cell differentiation, reflecting motor neurons over the course of 14 days of differentiation using 1 μM RA, 10X magnification.

[0036] Fig.11 Shown are NSC-34 cell differentiation, reflecting motor neurons at day 1, day 3, and day 5 of differentiation using 30 μM PGE2, 10X magnification.

[0037] Fig.12Shown is a bar graph reflecting the % viability of differentiated NSC 34 cells to different MLC901 concentrations.

[0038] Fig.13 Shown are day 7 NSC-34 cell differentiation studies using βIII tubulin and DAPI.

[0039] Fig.14 Shown are ICC staining using DAPI and βIII-tubulin in NSC-34 cells at day 3 after treatment with different concentrations of MLC901.

[0040] Fig.15 Shown is a bar graph reflecting the percent viability of NSC-34 cells in response to different MLC901 concentrations compared to untreated cells.

[0041] Fig.16 Protein signaling pathways and different protein markers involved in nerve regeneration are shown.

[0042] Fig.17 Shown are the expression of protein markers p-AKT, P-GSKβ, ATF-3, GAP43, eIF2B, and p-53 in untreated (UT) cells and cells treated with 1000 and 1200 μg / mL MLC901.

[0043] Fig.18 Shown is a Western blot analysis of the expression of proteins involved in the PI3K / AKT neuroregeneration pathway in untreated injured NSC34 cells and cells treated with MLC901 at concentrations of 1000 and 1200 μg / mL.

[0044] Fig.19 Shown are bar graphs reflecting the expression of PI3K / AKT pathway proteins phospho-Akt (Thr308), phospho-GSK-3β (Ser9), and ATF-3 using different MLC901 concentrations.

[0045] Fig. 20 Shown are bar graphs reflecting the expression of PI3K / AKT pathway proteins GAP-43, p-53, and eIF2β using different MLC901 concentrations.

[0046] Fig.21 A schematic diagram of the in vivo mechanical spinal cord injury model in adult SD rats using the calibrated forceps compression method is shown.

[0047] Fig. 22 The motor BBB scale is shown, reflecting joint movement, paw placement, and toe clearance scores.

[0048] Fig.23Shown are line graphs of motor BBB scores for control (healthy) rats, untreated rats, and rats treated with MLC901 3-28 days after mechanical spinal cord injury.

[0049] Fig.24 Shown is a line graph of wheel-running inhibition ability of control (healthy) rats, untreated rats, and rats treated with MLC901 after mechanical spinal cord injury.

[0050] Fig.25 Shown are line graphs of a) the time taken to walk a 0.5 m grid and b) the distance traveled across the grid in 60 seconds for control (healthy) rats, untreated rats, and rats treated with MLC901 after mechanical spinal cord injury.

[0051] Fig.26 Shown are line graphs of the inverted grid test for control (healthy) rats, untreated rats, and rats treated with MLC901 after mechanical spinal cord injury.

[0052] Fig. 27 Depicted are bar graphs reflecting electrophysiological somatosensory evoked potentials (SEPs) showing latency, duration, and amplitude in rats treated and untreated with MLC901 before and up to 4 weeks after mechanical spinal cord injury.

[0053] Fig.28 Shown are the morphologies of the extracted spinal cords of control (healthy) rats, untreated rats, and rats treated with MLC901 after mechanical spinal cord injury.

[0054] Fig.29 Shown are histopathological analyses of H and E staining of spinal cords extracted from control (healthy) rats, untreated rats, and rats treated with MLC901 after mechanical spinal cord injury.

[0055] Fig.30 Shown are H and E stained transverse histopathological analyses of spinal cords extracted from control (healthy) rats, untreated rats, and rats treated with MLC901 after mechanical spinal cord injury.

[0056] Fig.31 Shown are the timing of motor, electrophysiological, and sensory assessments following KA injection in untreated (UT) and treated (T) animals relative to control (H) animals.

[0057] Fig.32 a) shows the BBB scores of control (H) and injured rats (UT and T) before and after injury. The BBB scores of control (H) rats before injury were all 21. Compared with the BBB scores of T animals (n=5) after injury, the BBB scores of UT animals on days 7, 14, 21 and 28 after injury were significantly reduced (p<0.05*).

[0058] Fig.32 b) shows the distance covered by H, UT and T rats in 5 minutes. UT rats traveled significantly less distance on days 3, 7 and 14 (p<0.5)* and on days 21 and 28 (p<0.1)** compared to T rats. In each day of assessment, H rats traveled significantly longer distances (p, 0.001***) compared to UT and T rats (n=5).

[0059] Fig.33 Four motor assessments are shown, namely a) grid walking, b) grid distance traveled, c) wheel running and d) inverted grid. T rats showed significantly better motor results compared to UT rats (p<0.05*, p<0.01**) (n=5, data are expressed as mean±SD).

[0060] Fig.34 Shown are a) amplitude, b) duration and c) latency of three somatosensory evoked (SEP) assessments, i.e., UT, T and H rats before injury, on days 14 and 28 after injury. Amplitude differences between UT and T injured rats were observed on days 14 and 28 with p<0.05*significance. Also shown are the different waveforms of H, UT and T rats on day 14 after injury d). Data are expressed as mean ± SD and analyzed using one-way ANOVA.

[0061] Fig.35 H and E images of cross sections of H, UT and T rats 28 days after injury are shown. Images show H and E stained sections. Darker stained areas show hemorrhagic lesions (pink) containing red blood cell bags (orange arrows). Lesions (blue arrows) (progressive necrosis and cavitation) were also observed in UT and T sections. Inflammation in UT and T samples indicated different manifestations of red blood cells and neutrophils. Images were taken at 4x, 10x, 20x and 40x magnifications, with a scale bar of 500um (n=5).

[0062] Fig.36 Shown are H, UT and T rats a) Immunohistochemistry images of cross sections stained with antibodies for GAP43, GFAP and DAPI, and b) fluorescence intensity, showing the expression of GAP43 (motor neuron marker) as green fluorescence, GFAP (astrocytes and glial cells) as red fluorescence, and DAPI (nuclear staining) as blue fluorescence. Compared with the T injury group, the strong downregulation of GAP43 and GFAP in the UT group led to more motor nerve and astrocyte localization and damage. Images were taken by confocal microscopy at 10x magnification, and the scale bar is 1000um (n=5). Images were analyzed and compared by Image J software.

[0063] Fig.37 The mechanism of action of KA-induced excitotoxicity in neuronal cells is shown.

[0064] Fig.38 The anatomical location of the T13 thoracic spinal cord segment is shown in a dorsal top view of a SD rat.

[0065] Fig.39 Schematic diagram of the chemical injury model using kainic acid is shown, wherein a) shows marking of T11-T13 vertebrae, b) shows subcutaneous incision, c) shows exposure of muscle and spine, d) shows removal of muscle layer, e) shows insertion of syringe into the space between T12 and T13 vertebrae, f) shows syringe placed at 45°, g) shows slow perfusion of KA (0.1uL / min) and h) shows wound closure.

[0066] Fig.40 a) shows the expression of AKT, p-AKT, p-GSK3β, GAP43, p53, ATF-3 and elf2β detected by WB in C, UT and T rats after kainic acid excitotoxic injury. b) shows the histogram of the relative quantitative expression of AKT, p-AKT, p-GSK3β, GAP43, p53, ATF-3 and elf2β. Compared with UT rats, T rats had higher expression of AKT, GAP43, p-53 (p<0.05*), and decreased expression of p-GSK3β (*p=0.05). There was no significant difference in the expression of ATF-3 and elf2β between T and UT rats. Glossary

[0067] This section is intended to provide guidance on the interpretation of the words and phrases (and their grammatical variations where appropriate) set out below.

[0068] The term "neuronal growth" in the specification relates to the generally directional outgrowth of axons and dendrites. Neuronal growth is important in synapse formation or development.

[0069] Unless otherwise noted, the terms "comprising" and "comprise" and their grammatical variations are intended to represent "open" or "inclusive" language such that they include the recited elements but also permit the inclusion of additional non-recited elements.

[0070] As used herein, the term "about" as used in relation to a numerical value means, for example, about ±30%, about ±20%, about ±10%, about ±5% or about ±1% of the numerical value. If necessary, the word "about" may be omitted from the definition of the present invention.

[0071] The words "a", "an", and "the" are used to describe elements and components of the present invention. This is merely for convenience and to give a general understanding of the present invention. Unless the context clearly indicates otherwise, the description should be understood to include one or at least one, and the singular also includes the plural. Thus, for example, the term "agent" includes reference to a single agent as well as multiple agents (including mixtures of agents). It should also be noted that the term "or" is generally used in its sense that includes "and / or", unless the context clearly indicates otherwise.

[0072] As used herein, the term "in vivo" includes reference to the use of a whole living organism. This is in contrast to the term "in vitro", which does not use a whole living organism. The term "in vitro" should be understood to include, in particular, "ex vivo" use, in which cells, tissues, etc. that do not form part of a whole living organism (e.g., cells or tissues from cell or tissue culture, biopsies, dead organisms, etc.) can be used. Other non-limiting examples of "in vitro" involve the use of cell extracts or lysates.

[0073] As used herein, the term "extraction" includes reference to separation methods in which plant material (eg, chopped parts of a plant, whether fresh or dried) is contacted with a liquid solvent to transfer one or more components of the plant material to the solvent.

[0074] The terms "patient" and "subject" are used interchangeably herein, and these terms include reference to any person or non-human animal (preferably a mammal) for whom treatment with the present invention is desired. However, it is understood that "patient" or "subject" does not imply the presence of symptoms. As used herein, the term "mammal" refers to any member of the class of mammals, including but not limited to humans and non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cattle, sheep, pigs, goats and horses; domestic / companion animals, such as dogs and cats; laboratory animals (e.g., rabbits and rodents, such as mice, rats and guinea pigs, etc.). Preferably, the mammal is a human.

[0075] The term "treatment" includes any and all uses of remedying a disease state or symptom, preventing the establishment of a disease, or otherwise preventing, hindering, delaying or reversing the progression of a disease or other undesirable symptoms. Thus, "treatment" includes both prophylactic and therapeutic treatment.

[0076] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in the range format is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of the disclosed range. Therefore, the description of the range should be regarded as having explicitly disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, the description of a range such as 1 to 6 should be regarded as having explicitly disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numbers within the range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0077]

[00136] The terms "disease," "disorder," and "condition" are used interchangeably herein unless the context clearly indicates otherwise.

[0078] RA means retinoic acid.

[0079] PGE2 means prostaglandin E2.

[0080] DAPI means 4',6-diamidino-2-phenylindole.

[0081] The BBB scale refers to the Basso, Beattie and Bresnahan motor rating scale, which is widely used to test the behavioral consequences of spinal cord injury (SCI) in rats.

[0082] SEP test means somatosensory evoked potential test. SEP test studies the transmission of body sensations to the brain and how the brain receives these sensations.

[0083] H and E staining means hematoxylin and eosin staining, and is one of the main tissue stains used in histology.

[0084] ICC staining means immunocytochemistry staining and refers to staining of intact cells, either isolated or cultured, where the sample may be from adherent or suspension tissue culture cell lines.

[0085] ImageJ software is open source software for processing and analyzing scientific images.

[0086] MTT assay means mean transit time assay and is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity.

[0087] DPBS means Dulbecco's phosphate buffered saline.

[0088] DMEM / F12 means Dulbecco's modified Eagle's medium / Nutrient mixture F-12.

[0089] FBS means fetal bovine serum.

[0090] AA means amino acid.

[0091] NEAA means non-essential amino acids.

[0092] KA means kainic acid.

[0093] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. DETAILED DESCRIPTION

[0094] With reference to the following drawings, this article discloses in detail the detailed embodiments and implementation methods of the claimed subject matter with technical content, structural features, and the purpose and effect of realization. It should be understood that the disclosed embodiments and implementation methods are only illustrations of the claimed subject matter that can be embodied in various forms. However, the present disclosure can be embodied in many different forms and should not be construed as being limited to the exemplary embodiments and implementation methods described herein. On the contrary, these exemplary embodiments and implementation methods are provided to make the description of the present disclosure comprehensive and complete, and will fully convey the scope of the present disclosure to those skilled in the art. In particular, the terms in the embodiments of the present disclosure are only used for the purpose of describing a certain embodiment, rather than limiting the present disclosure. In the following description, the details of well-known features and technologies may be omitted to avoid unnecessarily obscuring the proposed embodiments and implementation methods.

[0095] The configurations discussed in the following description are non-limiting examples that may vary and are cited merely to illustrate at least one embodiment and are not intended to limit the scope thereof.

[0096] In one embodiment, a method of treating a subject to induce and / or accelerate recovery from spinal cord injury is provided, the method comprising administering to the subject an effective amount of an herbal composition comprising at least four herbal ingredients: Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong and Danggui (root of Angelica sinensis) or extracts thereof.

[0097] In one embodiment, the herbal composition of the method may further comprise at least one herbal ingredient selected from the group consisting of Danshen (Danshen root), Paeonia lactiflora (Paeonia lactiflora root), Carthamus tinctorius (Carthamus tinctorius), Prunus persica (Peach seed) and Acorus tatarinowii (Acorus calamus rhizome) or extracts thereof.

[0098] In one embodiment, the herbal composition of the method may consist essentially of at least four herbal ingredients or extracts thereof, the four herbal ingredients being Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong and Danggui (root of Angelica dahurica).

[0099] In one embodiment, the herbal composition of the method may consist essentially of at least nine herbal ingredients or extracts thereof, the nine herbal ingredients being Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong, Danggui (root of Danggui), Danshen (root of Danshen), Paeonia lactiflora (root of Paeonia lactiflora), Carthamus tinctorius (flower of Carthamus tinctorius), Prunus persica (seed of Peach) and Acorus tatarinowii (rhizome of Acorus calamus).

[0100] In one embodiment, the herbal composition of the method consists of a combination of extracts of nine herbal ingredients: Polygala tenuifolia, Astragalus membranaceus, Ligusticum chuanxiong, Angelica sinensis, Salvia miltiorrhiza, Paeonia lactiflora, Carthamus tinctorius flower, Persica persica, and Acorus tatarinowii.

[0101] In one embodiment, the herbal composition of the method is a pharmaceutical composition. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or excipients.

[0102] In a specific embodiment, the pharmaceutical composition used in the method herein comprises a combination of extracts of nine herbal ingredients, namely, Polygala tenuifolia, Astragalus membranaceus, Ligusticum chuanxiong, Angelica sinensis, Salvia miltiorrhiza, Paeonia lactiflora, Carthamus tinctorius, Semen persica and Acorus tatarinowii, and one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment, the pharmaceutical composition is MLC901 (also known as NeuroAiD II TM ).

[0103] In another specific embodiment, the pharmaceutical composition used in the method herein comprises a combination of extracts of four herbal ingredients, namely, Polygala tenuifolia, Astragalus membranaceus, Ligusticum chuanxiong and Angelica sinensis, and one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment, the pharmaceutical composition is MLC1501.

[0104] In one embodiment, recovery from spinal cord injury is induced and / or accelerated by neuronal cell regeneration.

[0105] In one embodiment, the herbal composition of the method promotes the growth of neuronal cells.

[0106] In one embodiment, the neuronal cell is a vertebrate neuronal cell. The vertebrate neuronal cell is a human neuronal cell. The neuronal cell is derived from a cortical neuronal cell.

[0107] In one embodiment, administration of the herbal composition results in regeneration of the spinal cord and / or results in regeneration of connections between the brain and the spinal cord and / or results in regeneration of damaged neuronal tissue or cells.

[0108] In one embodiment, administration of the herbal composition promotes recovery of limb movement in a paralyzed subject.

[0109] In one embodiment, the effective amount of the herbal composition is from about 1 mg / kg to about 100 mg / kg.

[0110] In one embodiment, a method is provided that includes administering a second agent for treating a subject having a spinal cord injury.

[0111] In one embodiment, the second agent is a drug effective for controlling pain and / or muscle spasm.

[0112] NeuroAid II TM , referred to herein as "MLC901," is a composition containing extracts of nine herbal ingredients (Astragalus, Radix salvia miltiorrhizae, Paeonia lactiflora, Rhizoma chuanxiong, Angelica sinensis, Carthamus tinctorius, Prunus persica, Polygala tenuifolia, and Acorus tatarinowii). It is currently marketed as an oral therapeutic to support recovery after stroke.

[0113] MLC1501 is a combination of extracts from four herbal ingredients (Astragalus, Chuanxiong, Polygala and Angelica) that is currently being evaluated in human clinical trials for stroke recovery.

[0114] MLC901 and MLC1501 and their preparation are described in published PCT application number WO 2017 / 048191A1, the contents of which are incorporated herein by reference in their entirety.

[0115] In one embodiment, the herbal composition of the present method consists essentially of four herbal ingredients (Polygala tenuifolia, Astragalus membranaceus, Ligusticum chuanxiong and Angelica sinensis) or extracts thereof and any 1, 2, 3, 4 or 5 of herbal ingredients (Danshen, Paeonia lactiflora, Carthamus tinctorius, Semen persica and Acorus calamus) or extracts thereof. Such herbal compositions and their preparation are described in published PCT application numbers WO 2007 / 106049 A1, WO 2010 / 053456 A1, WO 2010 / 110755 A1 and WO 2013 / 141818 A1, the contents of which are incorporated herein by reference in their entirety.

[0116] The pharmaceutical compositions of the present disclosure may optionally include one or more pharmaceutically acceptable additives, carriers, and / or diluents. Examples of pharmaceutically acceptable additives include pharmaceutically acceptable excipients, buffers, adjuvants, stabilizers, diluents, fillers, preservatives, lubricants, or other pharmaceutically acceptable materials known to those skilled in the art or as described herein. Examples of suitable pharmaceutical carriers or diluents include phosphate buffered saline solutions, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, and the like. Examples of excipients that may be used include, for example, sugars, starches, celluloses, gums, proteins, dextrins, and maltodextrins. A variety of formulations are well known and are discussed in depth in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton, Pennsylvania). In at least some embodiments, the pharmaceutical compositions as described herein include excipients such as dextrin or maltodextrin.

[0117] In one embodiment, the compositions of the present disclosure (e.g., pharmaceutical compositions) may be included within a kit. In addition to the herbal ingredients, the kit may also include instructions for use. The kit may be promoted, distributed, and / or sold as a unit for performing an aspect of the present disclosure.

[0118] Generally, the pharmaceutical compositions of the present disclosure may be prepared by methods known to those of ordinary skill in the art.

[0119] The composition may be, for example, a solution, suspension, liquid, chopped herbs, powder, paste (aqueous, non-aqueous), or any combination thereof.

[0120] The compositions of the present disclosure (e.g., pharmaceutical compositions) may be administered in liquid, semi-liquid, or solid form by any suitable route, such as orally, parenterally, intravenously, subcutaneously, intradermally, intraperitoneally, or topically, and formulated in a manner suitable for each route of administration. The term "administering" and variants of this term (including "administer" and "administration") include bringing the compositions of the present disclosure into contact with an organism or surface by any appropriate means, applying, delivering, or providing the compositions of the present disclosure to the organism or surface.

[0121] The herbal ingredients may be administered in a therapeutically effective amount (either as a single dose or as part of a series of doses). An "effective amount" or "therapeutically effective amount" means an amount administered to achieve a physiologically significant amount. A pharmaceutical agent has physiological significance if the amount of the agent causes a detectable change in the physiology of the recipient patient, resulting in a beneficial or desired outcome.

[0122] The exact amount required will vary from subject to subject, depending on factors such as the species being treated, the age, weight and general health of the subject, the condition being treated and the severity of the condition, mode of administration, sex of the subject, diet, time and frequency of administration, drug combination, and tolerance / response to therapy.

[0123] In one embodiment, the composition (e.g., pharmaceutical composition) of the present disclosure is administered as a capsule, taken once or more (e.g., 1, 2, 3, or 4 times) per day. In a specific embodiment, MLC901 is administered in the form of 2 capsules, taken 3 times per day. In a specific embodiment, MLC1501 is administered in the form of 4 capsules, taken 2 times per day. For patients with dysphagia, the capsule can be opened and the powder diluted in water that can be drunk like this or injected through a stomach tube.

[0124] The duration of treatment is usually 3 months or longer, which can be adjusted according to the patient's condition. In one embodiment, the patient's daily dose is about 500 mg to about 8 g, or about 1 g to about 8 g (e.g., about 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g or 8 g). A "daily dose" can be a single unit dose or multiple unit doses (tablets, capsules, etc.) taken on a given day. However, it should be understood that the dosage may vary according to the patient's needs and the severity of the condition being treated, etc.

[0125] In one embodiment, the treatment lasts for about 12 weeks. In another embodiment, the treatment lasts for about 24 weeks. In another embodiment, the treatment lasts for about 36 weeks. In another embodiment, the treatment lasts for about 48 weeks. In another embodiment, the treatment lasts for more than about 48 weeks.

[0126] In one embodiment, the herbal composition of the present disclosure (e.g., MLC901 or MLC1501) can be administered to SCI subjects in combination with other known SCI treatments, such as one or more additional active agents that effectively control pain and / or muscle spasms. One or more additional active agents can be administered at the same time (e.g., simultaneously) or at different times (e.g., sequentially) and in different time periods, which time periods can be separated or overlapped from each other. In one embodiment, there may be a synergistic effect. One or more additional active agents can be administered by the same or different routes as the herbal composition of the present disclosure. The one or more additional active agents used and the appropriate route of administration and dosage level will be known to those skilled in the art, or can be easily determined by those skilled in the art. Typically, as is well known in the medical field, the dosage regimen may depend on various factors, including the patient's body size, body surface area, age, specific compound to be administered, sex, time and route of administration, general health status, and other drugs administered simultaneously. Although individual needs vary, it is within the scope of those skilled in the art to determine the optimal range of the effective amount of each ingredient. Typically, the dosage of one or more additional active agents will be the same or similar to that administered when the medicament is used without the herbal composition of the present disclosure. When the herbal composition of the present disclosure is administered with one or more additional active agents, the one or more additional active agents may be provided in a composition or kit comprising the herbal composition of the present disclosure, or the one or more additional active agents may be provided separately (i.e., not as part of a composition or kit providing the herbal composition of the present disclosure).

[0127] In addition to the above embodiments, the present disclosure also includes the following specific embodiments: Embodiment 1: A herbal composition comprising at least four herbal ingredients or extracts thereof for use in treating a subject to induce and / or accelerate recovery from spinal cord injury, the four herbal ingredients being Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong and Danggui (root of Angelica sinensis). Embodiment 2: The herbal composition for use according to embodiment 1, wherein the herbal composition further comprises at least one herbal ingredient selected from the group consisting of Danshen (Danshen root), Paeonia lactiflora (Paeonia lactiflora root), Carthamus tinctorius (Carthamus tinctorius), Prunus persica (Peach seed) and Acorus tatarinowii (Acorus calamus rhizome) or extracts thereof. Embodiment 3: The herbal composition for use according to embodiment 1, wherein the herbal composition consists essentially of at least four herbal ingredients or extracts thereof, the four herbal ingredients being Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong and Danggui gui (root of Danggui gui). Embodiment 4:The herbal composition for use according to embodiment 1, wherein the herbal composition consists essentially of at least nine herbal ingredients or extracts thereof, and the nine herbal ingredients are Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong, Danggui (root of Danggui), Danshen (root of Danshen), Paeonia lactiflora (root of Paeonia lactiflora), Carthamus tinctorius (flower of Carthamus tinctorius), Prunus persica (seed of Peach) and Acorus tatarinowii (rhizome of Acorus calamus). Embodiment 5: A herbal composition for use according to any one of embodiments 1 to 4, wherein an extract of each of the herbal ingredients is used in the composition. Embodiment 6: The herbal composition for use according to any one of embodiments 1 to 5, wherein the herbal composition is a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers or excipients. Embodiment 7: The herbal composition for use according to embodiment 6, wherein the pharmaceutical composition is MLC901. Embodiment 8: The herbal composition for use according to embodiment 6, wherein the pharmaceutical composition is MLC1501. Embodiment 9: According to any one of embodiments 1 to 8, the herbal composition for use, wherein spinal cord injury recovery is induced and / or accelerated by neuronal cell regeneration. Embodiment 10: According to any one of embodiments 1 to 9, the herbal composition for use promotes the growth of neuronal cells. Embodiment 11: The herbal composition for use according to embodiment 9 or embodiment 10, wherein the neuronal cells are vertebrate neuronal cells. Embodiment 12: According to the herbal composition for use according to embodiment 11, wherein the vertebrate neuronal cells are human neuronal cells. Embodiment 13: According to any one of embodiments 9 to 12, the herbal composition for use, wherein the neuronal cells are derived from cortical neuronal cells. Embodiment 14: According to any one of embodiments 1 to 13, the herbal composition for use, wherein administration of the herbal composition results in spinal cord regeneration. Embodiment 15: According to any one of embodiments 1 to 14, the herbal composition for use, wherein administration of the herbal composition results in regeneration of connections between the brain and the spinal cord. Embodiment 16: According to any one of embodiments 1 to 15, the herbal composition for use, wherein the administration of the herbal composition results in the regeneration of damaged neuronal tissue or cells. Embodiment 17: According to any one of embodiments 1 to 16, the herbal composition for use, wherein the administration of the herbal composition promotes recovery of limb movement in a paralyzed subject. Embodiment 18: According to any one of embodiments 1 to 17, the herbal composition for use, wherein the effective amount of the herbal composition is from about 1 mg / kg to about 100 mg / kg. Embodiment 19: According to any one of embodiments 1 to 18, the herbal composition for use comprises administering a second agent for treating a subject suffering from spinal cord injury. Embodiment 20: The herbal composition for use according to embodiment 19, wherein the second agent is a drug effective for controlling pain and / or muscle spasm. Embodiment 21: A herbal composition comprising at least four herbal ingredients or extracts thereof for use in the preparation of a medicament for treating a subject to induce and / or accelerate recovery from spinal cord injury, the four herbal ingredients being Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong and Danggui (root of Angelica sinensis). Embodiment 22: The herbal composition for use according to embodiment 21, wherein the herbal composition further comprises at least one herbal ingredient selected from the group consisting of Danshen (Danshen root), Paeonia lactiflora (Paeonia lactiflora root), Carthamus tinctorius (Carthamus tinctorius), Prunus persica (Peach seed) and Acorus tatarinowii (Acorus calamus rhizome) or extracts thereof. Embodiment 23: The herbal composition for use according to embodiment 21, wherein the herbal composition consists essentially of at least four herbal ingredients or extracts thereof, the four herbal ingredients being Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong and Danggui gui (root of Angelica sinensis). Embodiment 24: The herbal composition for use according to embodiment 21, wherein the herbal composition consists essentially of at least nine herbal ingredients or extracts thereof, the nine herbal ingredients being Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong, Danggui (root of Danggui), Danshen (root of Danshen), Paeonia lactiflora (root of Paeonia lactiflora), Carthamus tinctorius (flower of Carthamus tinctorius), Prunus persica (seed of Peach) and Acorus tatarinowii (rhizome of Acorus calamus). Embodiment 25: According to any one of embodiments 21 to 24, an extract of each of the herbal ingredients is used in the composition. Embodiment 26: The herbal composition for use according to any one of embodiments 21 to 25, wherein the herbal composition is a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers or excipients. Embodiment 27: The herbal composition for use according to embodiment 26, wherein the pharmaceutical composition is MLC901. Embodiment 28: The herbal composition for use according to embodiment 26, wherein the pharmaceutical composition is MLC1501. Embodiment 29: According to any one of embodiments 21 to 28, the herbal composition for use, wherein spinal cord injury recovery is induced and / or accelerated by neuronal cell regeneration. Embodiment 30: According to any one of embodiments 21 to 29, the herbal composition for use promotes the growth of neuronal cells. Embodiment 31: The herbal composition for use according to embodiment 29 or embodiment 30, wherein the neuronal cells are vertebrate neuronal cells. Embodiment 32: According to the herbal composition for use according to embodiment 31, wherein the vertebrate neuronal cells are human neuronal cells. Embodiment 33: According to any one of embodiments 29 to 32, the herbal composition for use, wherein the neuronal cells are derived from cortical neuronal cells. Embodiment 34: According to any one of embodiments 21 to 33, the herbal composition for use, wherein administration of the herbal composition results in spinal cord regeneration. Embodiment 35: According to any one of embodiments 21 to 34, the herbal composition for use, wherein administration of the herbal composition results in regeneration of connections between the brain and the spinal cord. Embodiment 36: According to any one of embodiments 21 to 35, the herbal composition for use, wherein the administration of the herbal composition results in the regeneration of damaged neuronal tissue or cells. Embodiment 37: According to any one of embodiments 21 to 36, the herbal composition for use, wherein the administration of the herbal composition promotes recovery of limb movement in a paralyzed subject. Embodiment 38: According to any one of embodiments 21 to 37, the herbal composition for use, wherein the effective amount of the herbal composition is about 1 mg / kg to about 100 mg / kg. Embodiment 39: According to any one of embodiments 21 to 38, the method comprises administering a second agent for treating a subject with spinal cord injury. Embodiment 40:The herbal composition for use according to embodiment 39, wherein the second agent is a drug effective for controlling pain and / or muscle spasm.

[0128] In a further embodiment, the present disclosure provides an in vitro injury model of SCI using mechanical or chemical methods to cause injury in the NSC 34 cell line.

[0129] In a further embodiment, the in vitro mechanical injury method is Figure 1 and 2 Shown in.

[0130] In a further embodiment, the present disclosure has the following objectives: 1. To study the neurotoxic effect of the composition disclosed herein on NSC-34 cells. 2. Use NSC-34 cell line culture to create an in vitro SCI injury model. 3. Use time-lapse imaging and immunofluorescence labeling to determine the effects of the disclosed compositions on post-injury neurite regeneration in an in vitro SCI injury model. 4. Study potential protein signaling pathways in neuronal differentiation and regeneration when supplemented with the compositions of the present disclosure. In vitro mechanical injury model Objective 1: To study the neurotoxic effect of the disclosed composition on NSC-34 cells

[0131] method: NSC-34 cell line was cultured in DMEM / F12 (1: 1) + 10% FBS and 1% AA, and grown to 90% confluence, then treated with a differentiation medium consisting of DMEM / F12 (1: 1) + 1% FBS + 1% NEAA + 1% AA and 1 μM RA, 10 μM RA or 30 μM PGE2, and differentiated into mature motor neurons. Differentiation was determined by ImageJ and using differentiation markers βIII tubulin and DAPI to estimate the neurite growth from day 0 to day 14. Differentiated motor neurons were seeded into 96-well plates at a ratio of 5000 cells / well and attached for 24h. Then different concentrations of MLC901 (ranging from 25 μg / mL to 2000 μg / mL) were added to the wells and incubated for 24 and 48 hours. The neurotoxicity of MLC901 was assessed at a wavelength of 595nm using MTT salt assay and plate reader.

[0132] result: Using 1 μM RA, 10X magnification, Fig.10 The NSC-34 cell differentiation shown in reflects mature motor neurons at day 10 and day 14 of differentiation. Using 30 μM PGE2, 10X magnification, Fig.11 The NSC-34 cell differentiation shown reflects mature motor neurons at days 3 and 5 of differentiation. Fig.12 Bar graphs are shown reflecting the percent viability of differentiated NSC-34 cells to different MLC901 concentrations. MLC901 neurotoxicity results show the percent viability of differentiated NSC-34 cells to MLC901 concentrations ranging from 25 μg / ml to 2 mg / ml by MTT assay, with IC50 values ​​of 1178 μg / ml and 1251 μg / ml at 24 and 48 hours, respectively (n=6; results are shown as mean ± SD). Fig.13 Shown is a differentiation study of NSC-34 cells at day 7. The study used βIII tubulin and DAPI at 1 μM RA, 10 μM RA, and 30 μM PGE2. 10 μM RA and 30 μM PGE2 showed more cells with longer neurites (n=3).

[0133] discuss: Neurotoxicity studies using MLC901 gave an IC 50 value of 1251 μg / mL for differentiated NSC 34 cells. Therefore, a treatment concentration range of 800 μg / mL to 1400 μg / mL was selected for further study. In differentiation studies using different differentiation conditions (1 μM or 10 μM RA and 30 μM PGE2), it was found that 1 μM RA produced NSC-34 cell differentiation within 14 days, 10 μM RA produced NSC-34 cell differentiation within 7 days, and 30 μM PGE2 produced NSC-34 cell differentiation within 3 to 5 days. Therefore, 30 μM PGE2 was used for differentiation in in vitro mechanical injury studies. Objective 2: Create an in vitro spinal cord injury model using NSC-34 cell line culture

[0134] method: NSC-34 cell cultures were mechanically scratched using a pipette tip to produce two parallel vertical scratches. The cells were then washed three times with DPBS and observed under a microscope to examine damaged neuronal cells in the scratched area. Injured cells were treated with MLC901 at concentrations of 800, 1000, 1200, and 1400 μg / mL for 3 days, and neurite regeneration was estimated by neurite outgrowth and neurite count measurements in the injured area using ImageJ, time-lapse assays, and ICC staining. Figure 5 Bar graphs showing neurite outgrowth at different MLC901 concentrations on day 0 and day 3 are shown. Figure 6Shown are bar graphs of neurite counts at different MLC901 concentrations on day 0 and day 3. In these experiments, MLC901 concentrations of 1000 μg / mL and 1200 μg / mL showed longer neurite outgrowth than concentrations of 800 μg / mL and 1400 μg / mL (p>0.05) (n=6). Fig. 9 Bar graphs of neurite outgrowth after time-lapse imaging at day 0, day 1, day 2, and day 3 using different MLC901 concentrations are shown. Neurite outgrowth of untreated cells and cells treated with MLC901 at concentrations of 800 μg / mL, 1000 μg / mL, 1200 μg / mL, and 1400 μg / mL were displayed by ImageJ software. All treatment groups showed longer neurite outgrowth on day 3 compared to day 0 (p>0.05), and 800 and 1000 μg / mL showed significant (p<0.01) neurite outgrowth compared to UT cells and cells treated with 1200 μg / mL (p<0.001; n=6). Figure 5 , 6 In and 9, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.01.

[0135] result: Figure 4 Shown are the mechanical scratch injury model at day 0 and day 3 after treatment with different concentrations of MLC901, i.e., a) control cells with medium only, b) 800 μg / mL MLC901, c) 1000 μg / mL MLC901, d) 1200 μg / mL MLC901, and e) 1400 μg / mL MLC901. Figure 4 The line drawn in the figure indicates the mechanical injury area, and the arrow shows the neurite outgrowth, including the magnification of the selected area 10X, the scale bar is 100 μm. Compared with the untreated group, the treatment day 3 showed higher neurite outgrowth, and the MLC901 concentrations of 1000 and 1200 μg / mL provided the highest neurite outgrowth in the injury area (n=6). Fig.15 A bar graph is shown reflecting the percentage of viability of cells to different MLC901 concentrations compared to untreated cells. Here, MLC administered at 1200 μg / mL showed better cell viability (p>0.05) compared to untreated cells and cells treated with 800ug / mL and 1400ug / mL of MLC901 (n=3).

[0136] discuss:These results indicate that MLC901 treatment promotes neurite outgrowth in the injured area, especially at concentrations of 1000 μg / mL and 1200 μg / mL. It was also observed that neurite outgrowth in the treated group was superior to that in the untreated group. Therefore, the results clearly indicate that MLC901 promotes neurite outgrowth in healthy and injured cell cultures of differentiated NSC 34 cells. The MTT assay showed that the MLC901-treated injured cell group showed better viability percentage compared to the untreated group in both treated and untreated injured areas. Objective 3: To determine the effects of the disclosed compositions on the in vitro SCI injury model using time-lapse imaging and immunofluorescence labeling. Effects on neurite regeneration after injury

[0137] method: NSC-34 cell cultures were mechanically scratched using the above procedure and then treated with different concentrations of MLC901 (i.e., 800ug / mL, 1000μg / mL, 1200μg / mL, and 1400ug / mL) prepared in culture medium and placed in an incubator at 37°C. Neurite regeneration was observed using a camera over 72 hours, with photos taken every 30 minutes. At different points during the observation period, the regenerative capacity of MLC901 in the injured area was compared to untreated cells. After 3 days of treatment and time-lapse imaging, as shown in Figure 5. Figure 8 Cells were stained with βIII tubulin and DAPI to further visualize neurite regeneration as shown in Figure 1. The length of neurite outgrowth was measured at 20X magnification and statistically analyzed.

[0138] result: Figure 7 Time-lapse imaging is shown, indicating that MLC901 promoted neurite outgrowth and neurite counts in the injured area, with 1000ug / mL and 1200ug / mL showing greater neurite outgrowth in the injured area than other concentrations (n=3). Neurite outgrowth was quantified using ImageJ software. Figure 8 Shown are time-lapse imaging of neurite outgrowth from injured NSC34 cells at day 0 and day 3. Fig.14 Shown is ICC staining using DAPI and βIII tubulin at day 3 after treatment of NSC-34 cells with different concentrations (ie, 800 ug / mL, 1000 μg / mL, 1200 μg / mL, and 1400 ug / mL) of MLC901. Figure 4 The line drawn in the figure indicates the mechanical injury area, and the arrow shows the neurite outgrowth, including the magnification of the selected area 10X, the scale bar is 100 μm. Compared with the untreated group, the treatment day 3 showed higher neurite outgrowth, and the MLC901 concentrations of 1000 and 1200 μg / mL provided the highest neurite outgrowth in the injury area (n=6).

[0139] discuss: Time-lapse imaging showed that MLC901 promoted neurite outgrowth with longer neurites in the injured area when MLC901 was applied at concentrations of 1000μg / mL and 1200μg / mL compared to untreated cells. ICC staining after time-lapse imaging also demonstrated that βIII tubulin expression was greater after application of MLC901 at concentrations of 1000μg / mL and 1200μg / mL, indicating that these two concentrations were most beneficial for neurite regeneration. Objective 4: To investigate potential protein signals during neuronal differentiation and regeneration when supplemented with the composition of the present disclosure Transduction pathway

[0140] Methods and Results: like Fig.16 As shown in, the phosphoinositide 3-kinase (PI3K) / AKT signaling pathway contributes to a variety of processes, mediating many aspects of cellular function, including nutrient uptake, anabolic responses, cell growth, proliferation, and survival. The expression of different markers of this pathway indicates nerve regeneration. For example, the upregulation of p-AKT (Thr308) indicates regeneration. The downregulation of p-GSKβ3 (Ser9) reflects regeneration. Similarly, the increased expression of p53, ATF3, GAP43, and eIF2B (ser535) indicates neuronal regeneration. Fig.17 Western blot analysis of the expression of protein markers phospho-AKT (Thr308), phospho-GSKβ3 (Ser9), ATF-3, GAP43, eIF2β (ser535) and p-53 in untreated (UT) injured NSC34 cells and injured NSC34 cells treated with MLC901 at concentrations of 1000 μg / mL and 1200 μg / mL is shown. Compared with 1000 μg / mL MLC901, 1200 μg / mL MLC901 produces more protein marker bands, wherein 1200 μg / mL MLC901 shows less phospho-GSKβ3 (Ser9) expression compared with 1000 μg / mL MLC901, indicating that 1200 μg / mL MLC901 provides better nerve regeneration. Fig.18 Western blot analysis of the expression of proteins involved in the PI3K / AKT neuroregeneration pathway in injured NSC34 cells is shown. The cells examined consisted of untreated cells and cells treated with MLC901 at concentrations of 1000 μg / mL and 1200 μg / mL. Cells treated with MLC901 at a concentration of 1200 μg / mL showed the highest expression of neuroregeneration proteins. Fig.19It is a bar graph reflecting the 72h expression of PI3K / AKT pathway proteins a) phospho-Akt (Thr308), b) phospho-GSK3β (Ser9) and c) ATF-3 in different MLC901 concentration groups (i.e., untreated control, 1000 μg / mL and 1200 μg / mL). Compared to untreated cells, phospho-Akt (Thr308) and ATF-3 showed upregulation, while phospho-GSK3β (Ser9) showed statistically significant downregulation, indicating nerve regeneration. Fig. 20 Is a bar graph reflecting the 72h expression of PI3K / AKT pathway proteins d) GAP-43, e) p53 and f) eIF2β (ser535) in different MLC901 concentration groups (i.e., untreated control, 1000 μg / mL and 1200 μg / mL). Compared to untreated cells, eIF2β (ser535) showed statistically significant (p>0.01) upregulation at 1000 μg / mL and 1200 μg / mL, indicating regeneration.

[0141] discuss : Compared with untreated cells, the higher expression of markers in 1200 μg / mL MLC901 indicates more neurite regeneration. Similarly, the lower expression of phospho-GSK3β (Ser9) at 1200 μg / mL also indicates neuronal regeneration. These results provide support for the use of MLC901 as a neuroregenerative drug for the treatment of SCI.

[0142] In a further embodiment, the present disclosure provides an in vivo injury model of SCI in rats using mechanical and chemical methods.

[0143] In a further embodiment, the in vivo SCI injury method is shown in Figure 3a or 3b.

[0144] In a further embodiment, the present disclosure has the following objectives: 1. Establish an in vivo mechanical SCI injury model in adult rats. 2. To study the efficacy of the disclosed composition on neuronal regeneration in rats whose spinal cords were injured by mechanical means (eg, compression). 3. Establish an in vivo chemical SCI injury model in adult rats. 4. To study the efficacy of the disclosed composition on neuronal regeneration in rats whose spinal cords were damaged by chemical means (eg, using kainic acid).

[0145] In vivo spinal cord injury (SCI) models are considered irreplaceable as they can be related to similar pathophysiological conditions in humans. SCI models facilitate the understanding of injury mechanisms and the analysis of the benefits of advanced therapeutic interventions. The most commonly used animals for studying various neuronal pathological conditions are rodents, including rats and mice, due to their close similarities in the functional, morphological, and electrophysiological consequences of human SCI.

[0146] SCI is classified as complete or incomplete injury. Complete SCI describes the complete loss of sensation and muscle function at and below the injury site. Incomplete SCI refers to partial loss of function below the level of injury. The level of injury is another critical aspect: SCI in the cervical and upper thoracic regions can lead to inconsistent breathing patterns and cause death. Injury in the lower thoracic or lumbar regions is preferred as an SCI model because it only produces paraplegia without altering respiratory and cardiac function. SCI models are classified according to the mechanism of injury: mechanical or chemical. Mechanical injury is caused by mechanical means such as impactors, forceps, clamps, balloons or scissors, while chemical injury is caused by the injection of chemicals such as glutamate, aspartate, N-methyl-D-aspartate (NMDA), superoxide, hydroxyl radicals and pernitrates, heavy metals, ethidium bromide or kainate.

[0147] There are various mechanical injury models used to produce complete and incomplete SCI, such as contusion, compression, stretch, dislocation, or transection. Mechanical injury models are advantageous for assessing axonal regeneration and subsequent functional recovery. Chemical injury models can be used to study axonal and neuronal degeneration, molecular mechanisms, and the effects of various therapies on specific pathways. Excitotoxic chemical injury models are gaining popularity because they can be used to study secondary injury mechanistic events, such as neuronal and axonal degeneration caused by glutamate excitotoxicity. Neurodegeneration is described as the progressive loss of structure and function of neurons, axons, and nerve cells. Chronic neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, temporal lobe epilepsy, and amyotrophic lateral sclerosis (ALS) occur due to chemical excitotoxicity. 6-Hydroxydopamine (6-OHDA) causes neurotoxicity, leading to Parkinson's disease, and the G93A mutation causes the production of hydroxyl radicals in transgenic ALS rats, which further implicates oxidative damage in the pathogenesis of ALS. Exposure of the brain and spinal cord to other chemicals, such as heavy metals (e.g., aluminum), can lead to cognitive impairment and generally damage the nervous system, while scopolamine can cause dementia, colchicine can induce Alzheimer's disease symptoms, and kainic acid (KA) can produce temporal lobe epilepsy via intrahippocampal or intraamygdala administration or SCI via intraspinal administration.

[0148] Kainic acid (KA) is an agonist of ionotropic glutamate receptors that induces neuropathological changes in vivo and in vitro and is often used to study the mechanisms of excitotoxicity-induced neuronal apoptosis. Excitotoxicity plays an important role in many neurological disorders, including brain and spinal cord ischemia, trauma, and other neurodegenerative disorders. L-Glutamate is the main excitatory transmitter located in the nervous system. As a synaptic neurotransmitter, it induces long-term changes in synaptic organization, neuronal migration, neuronal excitability during the developmental stage and ensures neuronal viability. Excessive activation of glutamate receptors by KA alleviates the intracellular calcium influx, governs the production of free radicals, namely ROS (reactive oxygen species) and RNS (reactive nitrogen species), as well as ATPase, which triggers an additional influx of harmful ions and chemicals, leading to neuronal death. The administration of KA causes glutamate-mediated excitotoxicity, leading to neuronal death and neurodegeneration. The proposed mechanism of action of KA-induced excitotoxicity is described in Fig.37 Depicted in.

[0149] The use of KA-induced neuronal excitotoxicity models is a useful way to screen potential therapeutic drugs for nerve regeneration in SCI. The KA-induced SCI model in Sprague Dawley (SD) rats is described below. In this study, motor, electrophysiological, neurological and histological changes that occurred after treatment with MLC901 after intraspinal administration of KA were measured to evaluate the ability of MLC901 to induce nerve regeneration in SCI.

[0150] The tests used in the in vivo studies were as follows: Sports activities

[0151] (a) Open field test The open field test (OFT) was performed to evaluate changes in rat locomotor activity. Rats were placed in the middle section of an acrylic box, and two blinded observers observed the locomotor activity of each rat for 5 minutes. The rats were then scored according to the BBB scale. The bottom of the BBB scale was divided into three different sections. The inner square contained a distance of 20 cm, the middle square contained a distance of 40 cm, and the outer square contained a distance of 60 cm. The ground of the open field was marked with 10 cm x 10 cm boxes, and the number of boxes crossed during the 5-minute assessment period on days 3, 7, 14, 21, and 28 after injury was recorded and compared for all groups.

[0152] (b) Wheel test The wheel run is one of the most widely used tests for assessing motor deficits in rodents with brain and spinal cord injuries. In this test, rats are placed in a wheel, which is then forcibly rotated 90°. The rat attempts to inhibit the movement of the wheel by coordinating the forelimbs and hindlimbs to grasp the wheel, and the time it takes the rat to completely inhibit and stop the wheel is recorded for each rat in all groups. The scale used is as described below (0-4). 0 No inhibition 1 Poor inhibition 2 Slightly inhibited 3 Moderate inhibition 4 Complete inhibition

[0153] (c) Grid walking test An elevated metal square grid (40 x 60 cm) was used in this experiment [see Fig.33 c)]. The grid apparatus was placed in the open field apparatus described above. The rats were placed at one end of the grid and were allowed to walk and reach the other end of the grid. Walking behavior, such as the number of failures, total number of steps, and time spent walking the grid, was recorded and then analyzed by an experimenter who was blinded to the experimental design.

[0154] (d) Inverted grid test The inverted grid test is a test that assesses forelimb and hindlimb muscle strength. Normally, rats can easily grasp the grid in an inverted position for 30 to 40 seconds. Therefore, to assess strength after injury, the rats were placed upside down on a grid approximately 20 cm above the ground and a timer was set. The time each rat was able to grasp the grid in an inverted position was recorded. Sensory function test

[0155] (a) Hot scraper method The hot scraper test is used to evaluate changes in the sense of nociception in rats. The standard temperature is determined by placing a hot scraper on the tail of a control rat, slowly raising the temperature, and stopping when the rat begins to lick its tail. The average temperature calculated for all control rats is used as the standard temperature. The latency of temperature sensation (licking the tail) is recorded and compared by group. An increase in latency indicates a loss of sense of nociception, and the threshold for response to temperature increases. The stimulation duration does not exceed 20 seconds to avoid burns. Repeat the process three to five times, and take the average as the threshold. The baseline for normal rats is about 12 seconds. A 0-3 scale based on rat behavior is used as described below: 0 No withdrawal 1 Slow withdrawal 2 Delayed withdrawal 3 Rapid withdrawal and repeated flicking

[0156] (b) Cold sensation test The cold sensation test can also be used to test sensory function. Anhydrous ethanol (98%) is mainly used to test the cold sensation of rats. First, the target area of ​​the rat's skin is shaved. Then anhydrous ethanol is sprayed on the shaved area, and the reaction is recorded and scored and graded according to the same scale as above. Electrophysiological evaluation

[0157] Somatosensory evoked potential (SEP) measurement SEPs are responses in the brain and spinal cord that are elicited by movement and electrical stimulation of peripheral nerves. Commonly used stimulation sites in rats are the sciatic nerve at the wrist and the posterior tibial nerve at the ankle. Viking Quest TM SEPs were measured in all rats in each group. Rats were anesthetized, and then the stimulating electrode was fixed on the hind legs. The recording electrode was placed on the sensory area of ​​the hindlimb cortex between the coronal and sagittal sutures. The reference electrode was placed 0.5 cm behind the recording electrode, and a direct current square wave electrical stimulation with an intensity of 10–30 mA, a pulse width of 0.1 ms, and a frequency of 1 Hz was induced, superimposed 50–60 times. The SEP latency, duration, and amplitude were recorded, and the neurophysiological recovery of all groups was observed. Histological analysis after spinal cord extraction Four weeks after surgery and treatment, the rats were sacrificed. Spinal cord tissues (T12-T13) were dissected and fixed overnight in 4% paraformaldehyde, followed by 30% sucrose, and then cut into 15-mm thick sagittal and parasagittal sections using a cryostat. Hematoxylin-eosin (H&E) staining was performed for general histological examination under microscopic observation. Immunohistochemical analysis Immunohistochemistry was performed on 5 μm thin spinal cord tissue sections using specific antibodies. The sections were incubated overnight at 4 ° C in a humidified chamber in monoclonal GAP43 (D9C8) rabbit antibodies (1:200 cell signaling, USA) and GFAP monoclonal antibodies (1:200 ThermoFisher Scientific) and then stained for 2 h at 37 ° C with anti-rabbit IgG 488 and 594 secondary antibodies (1:400; Santa Cruz Biotechnology, Santa Cruz, California, USA). Nuclear staining was performed by using DAPI (1; 15000) in DPBS solution and incubated in the dark for 30 to 40 minutes at room temperature. Finally, slides were mounted and observed under a confocal microscope (Nikon A1R). In vivo injury model Objective 1: To establish an in vivo mechanical spinal cord injury model in adult rats (calibrated forceps compression method)

[0158] method: Calibrated forceps compression was performed by applying 15 seconds of compression on the T12 vertebra using forceps (Dumont #5). The arms of the forceps were placed on adjacent sides of the exposed spinal cord and compression was applied until the bases of the forceps tips touched each other. The forceps were held in this position for 15 seconds to cause moderate injury. Fig.21 The compression method is shown, where a) to h) represent the following: a) prone animal position, b) marking of T10, T12, and T13 vertebrae, c) subcutaneous incision, d) muscle removal, e) exposure of the spinal cord, f) removal of T12 vertebrae, g) compression of the spinal cord for 15 seconds, and h) wound closure (tissue and skin suturing). Objective 2: To study the effect of the disclosed composition on neurons of rats whose spinal cords were injured by mechanical means (e.g., compression) Regeneration effect

[0159] method: MLC901 was first administered subcutaneously using a single solution dose of 10 mg / kg of MLC901 in saline 30 minutes after mechanical induction of spinal cord injury in rats, and then orally administered at 10 mg / kg / day in drinking water for 4 weeks. If the animal does not take the drug orally, an oral gavage (20-16G, 3.8 to 10 cm) technique is used. The oral gavage needle is gently advanced along the palate until it reaches the esophagus. The tube should enter the esophagus easily. Once correct placement is confirmed, the drug is slowly administered through a syringe connected to the end of the needle. After administration, the needle is gently removed at the same angle as insertion. During treatment, motor function was assessed using the open field motor scale (BBB). Other tests include wheel running, grid walking, and inverted grid tests as discussed above. After treatment, the rats were killed (28 days later) and their spinal cords were extracted for further evaluation. Post-mortem evaluation may include electrophysiology (SEP and / or MEP), histology and immunohistochemistry, and testing for PI3K / Akt pathway neurite regeneration protein markers.

[0160] result: Sprague Dawley rats were divided into three groups: (i) SCI rats treated with MLC901, (ii) SCI rats that did not receive treatment, and (iii) healthy rats. The number of rats in each group was five. After injury using the above method, the animals were treated with 10 mg / kg / day of MLC901 in drinking water. If the animals were not medicated with water, the animals were force-fed by oral gavage (16G). The duration of treatment was 28 days, and during this 4-week period, the animals' motor movements were assessed by open field, running wheel, inverted grid, and electrophysiological analysis, such as Figure 23-27 The open field analysis was performed by two blinded individuals using Fig. 22 The paws were scored using the BBB scale shown in , reflecting subscores for joint motion, paw placement, and toe clearance. Fig.23 Line graphs of BBB scores and subscores for control (healthy rats), untreated rats, and treated rats after mechanical spinal cord injury at days 3, 7, 14, 21, and 28 after injury are shown. As demonstrated, treated rats showed better BBB scores in jaw movement, paw placement, and toe clearance compared to untreated rats (p<0.05). Fig.24 Shown is a line graph of wheel-running inhibition ability of control (healthy rats), untreated rats, and treated rats after mechanical spinal cord injury on days 3, 7, 14, 21, and 28 after injury on a scale of 0-4, where 0 represents no inhibition ability and 4 represents complete inhibition ability. As demonstrated, treated rats showed better inhibition ability compared to untreated rats (p<0.05), n=5. Fig.25 Line graphs of grid walking for control (healthy rats), untreated rats, and treated rats after mechanical spinal cord injury at days 3, 7, 14, 21, and 28 after injury are shown, where a) represents the time to walk a 0.5 m grid and b) is the grid distance traveled in 60 seconds (lines crossed). As demonstrated, the treated group showed better grid walking and more lines crossed compared to untreated rats (p<0.05).

[0161] Table 1 below shows the results of the grid walking test, including the total number of steps, the number of forelimb and hindlimb failures, and the total failure percentage in 1 minute for treated and untreated rats after mechanical spinal cord injury before injury and on days 3, 7, 14, 21, and 28 after injury. As demonstrated, the treated group showed statistically significant improved results (p<0.05*) compared to the untreated group on each day tested after injury. Table 1

[0162] Fig.26 Line graphs of the inverted grid test for control, untreated and treated rats after mechanical spinal cord injury before injury and at days 3, 7, 14, 21 and 28 after injury are shown, reflecting the time to grasp the grid in an inverted position, where the longer the time, the greater the ability and strength of the forelimbs and hindlimbs. As demonstrated, the treated group was able to maintain the inverted position for a longer time compared to untreated rats (p<0.05).

[0163] Table 2 below shows the results of the sensory function assessment of cold and heat reflexes, where sensory coordination tests were performed on treated and untreated rats using a cold and heat sensation / scale of 0-3, where 0 means no response, 1 means a partial response, 2 means a brief vocalization, and 3 means a continuous vocalization. Table 2

[0164] Fig. 27 It is a bar graph reflecting electrophysiological SEP, showing the latency, duration and amplitude of the treated and untreated groups. In the treated and untreated groups, the latency and duration increased, while the amplitude decreased. No significant difference was observed between the two groups.

[0165] Fig.28The morphology of the extracted spinal cord is shown. Fig.28 Shown in. Fig.29 and 30 Histopathological analysis of spinal cord lesions stained with H and E is shown.

[0166] discuss: The mechanical compression injury method successfully induced hindlimb paralysis in rats with moderate and incomplete SCI. The calibrated forceps compression method is a convenient and reproducible laboratory method in which the intensity of injury is controlled by the duration of compression. This method was used to perform gait analysis on MLC901-treated, untreated and healthy rats in multiple experiments. After the experiment, the MLC901-treated group recovered movement within 2 weeks, while the untreated group took 4 weeks to recover movement. The results of the wheel running, grid walking and inverted grid tests also showed that MLC901-treated rats scored significantly better than untreated rats, indicating that MLC901 helps restore lost connections between the brain and spinal cord. The SEP results showed a shorter latency and higher amplitude, indicating potential transmission disruption. Objective 3: Establish an in vivo chemical spinal cord injury model in adults

[0167] method: Fifteen (15) adult Sprague Dawley (SD) rats (weight 300g to 400g) were housed in clean cages under a biobubble air control system and maintained in a 12-hour light-dark cycle with free access to food and water. Seven days before the experiment, the rats were acclimatized. Rats were anesthetized with 0.1mL / 100g animal weight via intramuscular injection of ketamine-xylazine (9:1) solution. After the rats were anesthetized, ointment was applied to the eyes to prevent dehydration, and the rats were placed on a heating pad. The dorsal surface was shaved, and the vertebrae were marked from the T11-T13 position at about 1cm around the expected incision position. The T13 vertebra was determined by palpating the 13th rib from the outside, and then the vertebra was visualized with forceps. The forceps were gently hooked under the rib to determine which vertebra it was connected to, i.e., the T13 vertebra would move with the movement of the ribs. By counting upward, the T11 vertebra was determined. The incision site was disinfected three times with 70% isopropyl alcohol and then with iodine solution. Fig.38 The anatomical location of the T13 thoracic spinal cord segment is depicted in a dorsal view of a SD rat. The skin is opened to expose the spine at T12-L3. T13 is adjacent to the 13th rib.

[0168] Before making the incision, check the rat's reflexes using a toe or tail pinch to ensure they are properly anesthetized. Then, make an incision along the dorsal spine and check the rat's reflexes again. Draw a line along the T11-T13 vertebrae and incise the skin approximately 1.5 cm. Insert a retractor to secure the skin and clear tissue on both sides of the spinal cord to locate the T13 vertebra. Under proper lighting, identify the space between the T12 and T13 vertebrae. Slowly insert the needle between the vertebrae and check the reflexes of the lower extremities and tail for proper positioning.

[0169] KA (1 mL, 0.1 mM) was diluted with 1 mL of normal saline to produce a final concentration of 0.05 mM KA, of which 40 μL was injected at a rate of 0.01 mL (10 μL) / min until the syringe was empty. The rapid onset of leg and tail stiffness after injection indicated successful injury induction. After KA injection, the rat was placed in an upright position for 3-5 min to prevent drug infusion into the brain and then gently placed in a supine position on a heating pad. Then, gentle pressure was applied with a surgical sponge to stop bleeding, taking care not to apply pressure to the spinal cord.

[0170] The muscle layer above the spinal cord was carefully sutured, taking care not to damage or apply pressure to the spinal cord. The skin above the wound was closed using sutures, and the rat was placed on a ventilator and heating pad until consciousness was regained (60-90 minutes). Subsequently, 3–4 mL of lactated Ringer's solution / 300-400 g body weight (preheated to 37°C) was injected to avoid surgical dehydration. Tramadol stock solution (0.4 mg / 100 g, 50 mg / mL) was administered 5 minutes after wound closure. Povidone and topical antibacterial ointment were applied to the wound. The rat was closely monitored until consciousness was regained and then transferred to a regular clean cage with comfortable clean bedding. Fig.40 A schematic diagram of KA-induced spinal cord injury is provided, wherein: A) shows labeling of T11–T13 vertebrae; B) shows the subcutaneous incision; C) shows exposure of the muscle and spine; D) shows removal of the muscle layer; E) shows insertion of a 26-G syringe into the intervertebral space between T12 and T13 vertebrae; F) shows the syringe placed at a 45° angle; G) shows slow perfusion of KA (0.1 μL / min); and H) shows wound closure.

[0171] After spinal cord injury, tramadol 0.4 mg / 100 g was administered subcutaneously twice daily for 3–5 days to relieve pain symptoms. Soft food and autoclaved clean water were provided nearby and easily accessible to the rats. Daily food and water intake was carefully monitored, and if the rats were not eating and drinking properly, lactated Ringer's solution (1 mL / 100 g body weight subcutaneously) was administered 3–5 days after injury. Urinary retention usually occurs due to paralysis of the lower limbs. To avoid this, the bladder was manually massaged twice daily to promote urination: the abdomen was gently palpated to locate the bladder, and then gentle downward pressure was applied until the bladder was empty. If hematuria occurred, the antibiotic Baytril (100 mg / mL) was injected subcutaneously at 50 mg / 100 g body weight.

[0172] Injury was induced in two of the three groups of rats. After injury, the rats were divided into a treated group (T), an untreated group (UT), and a healthy group (C). The experimental time frame for various evaluation procedures was Fig.31 All rats were pre-evaluated on days 1, 3, and 7 one week before surgery. After surgery, electrophysiological evaluations were performed on days 0, 14, and 28, while motor tests were performed on days 3, 7, 14, 21, and 28. Sensory function tests were performed on days 7 and 21. The animals were sacrificed on day 28 to collect spinal cord specimens for histological and pathological studies. Objective 4: To study the efficacy of the disclosed composition on neuronal regeneration in rats with chemically injured spinal cord

[0173] method: The experimental time frame for various evaluations is Fig.31 All rats were pre-evaluated on days 1, 3, and 7 (1 week before surgery). After surgery, electrophysiological evaluations were performed on days 0, 14, and 28, while motor tests were performed on days 3, 7, 14, 21, and 28. Sensory function tests were performed on days 7 and 21. The animals were sacrificed on day 28 and the spinal cord specimens were taken for histological and pathological studies.

[0174] result: Motor activity assessment

[0175] After KA injury, rats exhibited complete paraplegia, no activity of the hind limbs or tail, and urinary dysfunction without defecation dysfunction. In both injury groups (T and UT), puncture retractions began to heal one week after injury. In the T group, slight hind limb movement occurred one week after injury, which became more obvious after 3 weeks. However, in the UT group, slight coordinated movement was observed only after day 14. Even after day 28, fully coordinated movement was not seen in either group. In the T group, the hind limbs showed slightly more coordinated activity after day 28, but less improvement was observed in the UT group. Fig.32a)]. After 3 days, urinary function was partially restored, but residual urine remained in the bladder in both injury groups and completely resolved after 7-10 days. The BBB scale contains the following eight categories, allowing for separate assessment of the forelimbs and hindlimbs: joint movement of the affected limb, weight support, toe position, paw placement, direction and movement when walking, limb coordination, and tail position. The maximum total score of 21 points indicates normal movement or complete functional recovery, and 0 indicates no movement. The BBB scores of the C, T, and UT groups were scored by two blinded observers. The results showed that the BBB scores of the UT injury group were significantly lower than those of the T group on days 7, 14, 21, and 28 after injury. Fig.32 a)].

[0176] The distance covered by rats before and after SCI was observed to examine spontaneous motor activity [ Fig.32 b)]. Recording and scoring were performed during a 5-minute walk in the open field test. The distance covered was significantly reduced in UT-injured rats compared to the T group. The calculated distances covered by UT and T rats on days 3, 7, and 14 were 10.5±0.707cm and 17.5±0.76cm (p<0.05*), 17.5±0.707cm and 25±2.707cm (p<0.05*), and 32.5±2.12cm and 39.2±3.53cm (p<0.05*). The distances covered by the UT and T groups on days 21 and 28 (p<0.01**) were 35±2.82cm and 47±1.12cm and 41±2.207 and 55±2.432, respectively. The reduction in distance covered by UT rats was matched by hindlimb paraplegia and loss of coordination[ Fig.32 a) and 32b)].

[0177] Locomotor gait analysis was also assessed in the open field. The locomotion of KA-injured rats was compared with that of control (healthy) rats and scored using the BBB scale by two blinded observers before injury and on days 7, 14, 21, and 28 after injury. Jaw movement, jaw placement, and toe clearance were scored in Fig.39 Shown in.

[0178] Wheel-running assessments were evaluated and compared between the C, UT, and T groups before injury. UT and T rats showed a complete loss of inhibition after injury and showed significantly reduced inhibition compared to the C group (p<0.001***). However, the T group showed greater inhibition than the UT group on days 3 and 14 (p<0.05) and days 21 and 28 (p<0.01). Fig.33 a)]. Similarly, grid walking and inverted grid assessments showed that both injury groups (UT and T) had a significant loss of strength to grasp the grid in an inverted position due to hindlimb paralysis and were unable to walk on the grid without significant foot failure [ Fig.33b), 33c) and 33d)]. However, as the treatment days progressed, the T group showed improvement (p<0.05 and p<0.01), as evidenced by longer hold times and grid distance traveled than the UT group. This was statistically significant at days 7, 14, 21 and 28 after injury ( Fig.33 ). Sensory function test

[0179] Sensory function was performed on days 7 and 21 after KA injury. The heat and cold sensation tests showed that after injury, both UT and T group rats showed no response on day 3, while on day 7, UT rats were scored as 0 by two observers, while T group rats showed slow withdrawal and were scored as 1 (see Table 3 below). The results showed delayed responses and indicated impaired sensory nerve conduction. However, before injury and C group rats scored 3 points throughout the experiment and withdrew quickly. The withdrawal response latency (time from stimulation to withdrawal of the hind paw) of KA injured rats was shorter than that of C group rats. Starting from day 7, it was observed that the withdrawal response latency of T rats was better than that of UT rats (see Table 3). Table 3 Electrophysiological evaluation

[0180] Results of the SEP test showed that the waveform disappeared immediately after KA-induced SCI in both injury groups. The electrophysiological results were correlated with the motor assessment results. The SEP waveform in the healthy group did not change significantly over time. In contrast, in the KA injury groups (UT and T), the SEP waveform disappeared immediately after SCI, and no waveform was observed even 30 minutes after the injury. Fig.34 a), b), c), and d) show the waveforms, mean onset amplitude, duration, and latency of the UT and T groups on days 14 and 28, respectively. A decrease in amplitude was observed in the UT and T injury groups compared with before injury and group C. The mean onset amplitude in the combined KA injury group was 19.56 ± 1.52 mV on day 14 and 22.3 ± 0.81 mV on day 28. The mean onset amplitude of group C on days 0, 14, and 28 was 25.63 ± 0.81 mV, 25.67 ± 0.50 mV, and 26.02 ± 1.14 mV [ Fig.34 b)]. The UT injury group showed a statistically (p<0.05) reduced amplitude on day 14 compared with the T group and a statistically (p<0.01) reduced amplitude on day 14 compared with the C group. Fig.34 b)]. The duration of nerve conduction indicates the nerve conduction velocity. It was observed that the duration of nerve conduction in the UT group (1.32±0.11ms) on day 14 was significantly increased compared with that in the T group (0.42±0.17ms) (p<0.05) [ Fig.34a) and c)]. Similarly, a statistically significant increase in latency was observed in the UT group after day 14 post-injury compared with the T group (p < 0.05). No somatosensory evoked potential waveform was observed just after day 0 of injury. However, after day 14, the UT rats showed severe motor dysfunction in the hindlimbs, as reflected by a sharp decrease in amplitude and a significant increase in duration and latency compared with the T group [ Fig.34 a), b), c) and d)]. Histological analysis

[0181] Tissue damage was observed in cross-sections of the spinal cord of rats with KA injury (UT and T). Histopathological studies of spinal cord tissue using H and E staining showed lesions at the site of KA injection. The most severe damage was in the center, with loss of the ventral horns of gray matter and the lateral cords of white matter (see Fig.35 ). H and E staining results showed that the structures of the UT and T groups were severely damaged and the number of neuronal cells was reduced compared with the C group. The presence of hemorrhagic foci was observed in the middle of the gray matter. The lesion area in the middle (gray matter) showed progressive necrosis and cavitation. Many red blood cells and neutrophils appeared in the primary lesion sites of both UT and T rats. Compared with T rats, UT rats showed larger hemorrhagic foci, which indicated more damage and neurodegeneration. Significant albumin extravasation was observed in the spinal cord sections of UT and T KA-injured rats. However, no albumin extravasation was observed in the C group. The presence of albumin extravasation in KA-injured rats is mainly caused by nervous system degeneration and glutamate excitotoxicity. This co-localization effect of albumin extravasation in neuronal cells suggests that neurotoxicity and neuronal cell death are more in UT rats than in T rats (see Fig.35 ). Immunohistochemical analysis

[0182] Macroscopic evaluation of the dissected spinal cord revealed significant hemorrhage and tissue damage at the lesion site in the KA injury group. Immunohistochemistry showed systemic expression of GAP-43 and GFAP in the UT and T groups. GAP43 expression was highly downregulated in the UT and T groups, revealing a close association with lesion formation and neuronal degeneration (see Fig.36 ). GAP-43 positive neuronal fibers were found in the gray matter of the C group. However, the expression of green fluorescence in the gray matter of the UT group was lower compared with the T group, reflecting neuronal degeneration. Double staining of GFAP also showed the appearance of astrocytes in the C, UT, and T groups. Lower GFAP expression also indicates astrocyte loss. Confocal images reflected significant differences in GAP-43 and GFAP expression between the C, UT, and T groups (see Fig.36 ).

[0183] discussAfter intraspinal administration of KA, male Sprague-Dawley rats showed cardinal signs of motor dysfunction and sensory impairment. Behavioral changes such as anxiety and a tendency to sit in the corner of the cage all the time were observed in KA-lesioned rats, mainly due to glutamate excitotoxicity. Rats that remained still for more than 15-20 seconds were forced to move by gently tapping or scratching the sides of the open field. If the animal still did not respond to these maneuvers, it was picked up and placed in the center of the open field apparatus, which caused it to move to the other side. Sometimes, the observation period was extended to more than 5 minutes to allow for a more accurate assessment of toe clearance, paw position, or forelimb-hindlimb coordination.

[0184] The BBB scale is an excellent tool to understand motor activity. The lowest score, "0, no observable hindlimb (HL) movement," is usually given in severe SCI cases. The UT rats in this study showed the lowest BBB score of 3 to 4 points, indicating moderate SCI. The T rat group reached 19 points on day 28 after injury. Two blinded observers scored the following behaviors: from single joint movements of the hindlimbs to plantar walking, to coordinated walking, and finally more subtle motor behaviors (such as paw position, trunk stability, and tail position). The highest score in the BBB scale is 21, indicating "consistent plantar walking and coordinated gait," revealing a fully recovered and balanced gait. Rats in the UT and T groups reached a maximum of 13 and 19 points (on day 28 after injury), reflecting motor dysfunction. Only the C group and pre-injury rats scored 21 points on the BBB scale. It was also observed that there was little improvement in the scores of UT rats between days 21 and 28 after ensuring injury. Rats in the T group showed a short lag period in the first three days after injury, followed by a faster recovery period between days 4 and 13, and then a functional plateau between days 15 and 28. The BBB score is important for interpreting changes in motor activity after SCI and was used in this paper to detect changes in results between the UT and T groups.

[0185] Rats placed in a running wheel tend to inhibit and stop the wheel by forceful grasping of the forepaws and hindpaws, which is why the running wheel test is considered valuable in assessing strength and coordination in rats after SCI. The BBB scores and distance covered (in cm) for all three groups showed a statistically significant (p<0.05* and p<0.01**) decrease in the scores of KA-injured rats, reflecting the successful induction of moderate paraplegia. The results also showed that T rats showed superior BBB scores and distance covered than the UT group, emphasizing the value of this model for studying the regenerative potential of therapeutic agents under laboratory conditions.

[0186] Likewise, the grid walking test is another important laboratory test used to evaluate and compare gait in different groups of rats and to study the regenerative potential of therapeutic agents under laboratory conditions. In the grid walking test, rats are required to place their limbs accurately in certain places. When a rat's paw falls off, this is considered a foot fault. Errors and faults are counted during walking on the grid, with more faults reflecting greater motor coordination deficits.

[0187] The results obtained from the wheel-running and grid-walking tests showed that the T group rats showed significantly better motor coordination than the UT group rats. This finding suggests that the motor control of the T group rats was substantially improved compared with that of the UT group rats [see Fig.33 a), b), c) and d)]. In the inverted grid test, the rats in group T showed significantly better ability than those in group UT.

[0188] The results showed that coordination and motor deficits associated with KA injury in rats persisted until day 28 after KA injury. For both UT and T group rats, these motor deficits remained unchanged until day 3 after injury, at which time T group rats subsequently began to show more improvement than UT rats until day 28, indicating that T group rats have neuroregenerative potential. The results obtained from all motor tests showed that MLC901 improved motor recovery in injured rats, which may also be related to increased GAP43 expression (neuronal marker) and reduced demyelination (H and E). GAP43 expression was higher in T rats compared to UT rats, indicating that MLC901 attenuated the improvement of motor activity by restoring lost connections and neuronal regeneration.

[0189] Electrophysiological assessment is reflected as a functional means of assessing the integrity of various aspects of the nervous system, including the spinal cord. Clinical applications of electrophysiology, particularly evoked potentials, such as somatosensory and motor evoked potentials, have been incorporated into the diagnosis of peripheral or central nervous system injuries, particularly somatosensory evoked potentials (SEPs) that monitor the integrity of the dorsal columns of the spinal cord [Cruccu et al., Clinical Neurophysiology, 119(8), pp. 1705–1719. (2008)]. SEPs provide a reproducible, non-invasive, and objective assessment of axonal conduction of descending sensory and motor pathways in the normal and injured spinal cord [Malhotra and Shaffrey, Spine, 35(25), pp. 2167–2179 (2010)]. Impulse conduction through a neuron can be measured by stimulating an electrical signal from the nerve itself or from a muscle innervated by that nerve. Latency is measured as the time from stimulation by the stimulating electrode to the deflection of the signal recorded by the recording electrode. The time it takes for an electrical impulse to propagate from the stimulation site to the recording site is called latency. The time required for conduction is called duration and is measured in milliseconds (ms). The size of the response to impulse conduction in motor and sensory neurons is called amplitude and is measured in millivolts (mv). The signal is recorded as a nerve conduction signal passing under the recording electrode, and the latency divided by the distance can estimate the conduction velocity (meters per second). The measurement can be described in terms of latency to the onset of the response, the amplitude of the response, and the conduction velocity (duration). In this study, the SEP waveforms of each group were observed in terms of latency, amplitude, and duration by placing electrodes in the sciatic nerve near the knee area of ​​rats. The increase in latency and duration in the two injury groups (T and UT) reflects demyelination of peripheral neurons and neuronal loss. Neuronal loss affects the amplitude of nerve conduction velocity. Neuronal demyelination leads to a slowing of latency. In the present study, both UT and T injury groups showed no waveform just after injury (within 30 min), but the latency of the T group increased significantly (p<0.05) on the 14th day after injury compared with the UT group. This indicates that nerve conduction improved after treatment. The decrease in amplitude is directly related to the presence of low motor neurons (neurodegeneration) and demyelinating lesions involved in impulse conduction. Low amplitude and increased latency and duration lead to slow nerve conduction velocity (NCV), which indicates demyelinating lesions, while low amplitude values ​​reflect axonal degeneration. In the present study, we found that NCV was not observed just after injury (30 min). However, on the 14th day, rats in the T group showed a better NCV than rats in the UT group.

[0190] Morphological and pathological interventions are also important in the evaluation of neurodegenerative disorders. Conventional H and E staining can show the corresponding structures of the spinal cord after injury and can also detect neural demyelination in spinal cord sections after SCI. H and E stained sections after KA injection show the presence of hemorrhagic lesions, especially in the gray matter. Progressive necrosis and cavitation were also observed in UT and T KA-injured rats (see Fig.35 However, hemorrhage and cavitation were more obvious in the UT group than in the T group, in which cavitation was reduced after MLC901 treatment ( Fig.35 ). There were more inflammatory cells (such as erythrocytes and neutrophils) in the UT and T groups compared with those observed in the C group, indicating that KA excitotoxicity-induced neurodegeneration ( Fig.35 ).

[0191] The expression of GAP43 and GFAP was determined to ensure the quality of neurons in all groups (i.e., C, T, and UT). Growth-associated protein 43 (GAP43) is an activity-dependent plasticity protein enriched in axons and neurons, which promotes actin polymerization and axon regeneration. Glial fibrillary acidic protein (GFAP) is the main structural protein of filaments within the cytoskeleton of astrocytes and serves as a marker for mature astrocytes. The results obtained showed that GAP43 was highly expressed in the C group, but less expressed in the injury group. The present study showed that GAP43-positive fibers were significantly increased in the T group (see Fig.36 ), indicating that MLC901 treatment may support neuronal and axonal regeneration. It was also observed that GFAP expression levels were higher in the T group compared with the UT group, supporting the fact that MLC901 treatment can protect against astrocyte loss ( Fig.36 ). However, greater expression of GFAP staining in the gray matter indicated the presence of glial and astrocytic scarring in the UT and T groups ( Fig.36 ).

[0192] In summary, the neurodegenerative outcomes of KA-induced injury and neuroregeneration in MLC901-treated rats were observed by motor assessment (i.e., open field test, wheel running, grid walking, and inverted grid), assessment of sensory function and SEP. The results found after H and E and IHC staining evaluated the presence of pathological features. All of these results demonstrate the success of the KA excitotoxicity model as an in vivo model for creating and studying SCI. In addition, the neuroregenerative potential of MLC901 was demonstrated by improvements in all motor activity scores, sensory, electrophysiological, and histological assessments. in conclusion

[0193] The in vivo KA-induced excitotoxicity model provides a practical, convenient, and effective method to induce SCI in laboratory animals. All the important findings of this study support the existence of the most prominent physiological and pathological consequences of SCI. Intraspinal administration of KA ameliorated the negative effects of the injury, resulting in incomplete paraplegia in rats. The main mechanism of action of KA excitotoxicity is mediated by activation of glutamate receptors, which lead to more Ca +2 The influx of MLC901 into nerve cells leads to the formation of ROS and RNS, which ultimately stimulates demyelination and neurodegeneration. Importantly, this study showed that treatment of KA-injured rats with MLC901 improved the functional recovery process of SCI relative to untreated (UT) rats. References

[0194] All references, articles, publications, patents, patent publications and patent applications ("references") cited herein are incorporated by reference in their entirety for all purposes. However, mention of any reference does not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. The references include the following: a) WO 2017 / 048191 A1 b) WO 2007 / 106049 A1 c) WO 2010 / 053456 A1 d) WO 2010 / 110755 A1 e) WO 2013 / 141818 A1 f)Cruccu et al., Clinical Neurophysiology, 119(8), pages 1705–1719.(2008) g)Malhotra&Shaffrey,Spine,35(25),pages 2167–2179(2010)

Claims

1. A method of treating a subject to induce and / or accelerate recovery from spinal cord injury, the method comprising administering to the subject an effective amount of an herbal composition comprising at least four herbal ingredients: Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong and Danggui (root of Angelica dahurica) or extracts thereof.

2. The method of claim 1, wherein the herbal composition further comprises at least one herbal ingredient selected from the group consisting of Danshen (Danshen root), Paeonia lactiflora (Paeonia lactiflora root), Carthamus tinctorius (Carthamus tinctorius), Prunus persica (Peach seed) and Acorus tatarinowii (Acorus calamus rhizome) or extracts thereof.

3. The method as claimed in claim 1, wherein the herbal composition consists essentially of at least four herbal ingredients or extracts thereof, the four herbal ingredients being Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong and Danggui (root of Angelica dahurica).

4. The method of claim 2, wherein the herbal composition consists essentially of at least nine herbal ingredients or extracts thereof, the nine herbal ingredients being Polygala tenuifolia (root of Polygala tenuifolia), Astragalus membranaceus (root of Astragalus membranaceus), Ligusticum chuanxiong, Danggui (root of Danggui), Danshen (root of Danshen), Paeonia lactiflora (root of Paeonia lactiflora), Carthamus tinctorius (flower of Carthamus tinctorius), Prunus persica (seed of Peach) and Acorus tatarinowii (rhizome of Acorus calamus).

5. The method of any one of claims 1 to 4, wherein the herbal composition is a pharmaceutical composition.

6. The method of claim 5, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

7. The method of claim 6, wherein the pharmaceutical composition is composition MLC901.

8. The method of claim 6, wherein the pharmaceutical composition is composition MLC1501.

9. A method as claimed in any preceding claim, wherein recovery from spinal cord injury is induced and / or accelerated by neuronal cell regeneration.

10. The method of any preceding claim, wherein the herbal composition promotes the growth of neuronal cells.

11. The method of claim 9 or claim 10, wherein the neuronal cells are vertebrate neuronal cells.

12. The method of claim 11, wherein the vertebrate neuronal cells are human neuronal cells.

13. The method of any one of claims 9 to 12, wherein the neuronal cells are derived from cortical neuronal cells.

14. The method of any preceding claim, wherein administration of the herbal composition results in spinal cord regeneration.

15. The method of any preceding claim, wherein administration of the herbal composition results in regeneration of connections between the brain and spinal cord.

16. The method of any preceding claim, wherein administration of the herbal composition results in regeneration of damaged neuronal tissue or cells.

17. The method of any preceding claim, wherein administration of the herbal composition promotes recovery of limb movement in a paralyzed subject.

18. The method of any preceding claim, wherein the effective amount of the herbal composition is from about 1 mg / kg to about 100 mg / kg.

19. The method of any preceding claim, wherein the method comprises administering a second agent for treating a subject suffering from spinal cord injury.

20. The method of claim 19, wherein the second agent is a drug effective for controlling pain and / or muscle spasm.

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