urokinase-type-plasminogen-activator-induced-mouse-back-pain-model

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

University of New Mexico Health Sciences Center

The blood–brain barrier (BBB) and blood–nerve barrier ensure protection of the nervous system but pose a challenge for the treatment of pain since it restricts passage of many therapeutic drugs. Although it is unknown which blood–neural barrier is more relevant, or whether permeabilities are the same for different barriers, we proposed that the inefficiency of thiazolidinedione-type agonists for peroxisome proliferator-activated receptor gamma (PPARɣ) is due to their difficulty in passage through the BBB. We developed a new highly BBB penetrable PPARɣ agonist for the treatment of neuropathic pain, assuming BBB permeability is a rule of thumb to estimate the overall permeability of relevant blood–neural barriers.  As an index of brain penetration, the brain–plasma ratio (Kp) of ELB00824 is 5.13, suggesting very high brain bioavailability, which is 58-fold that of pioglitazone. The series of studies presented here indicate that ELB00824 may be the most potent PPARɣ agonist currently known for acute reduction of neuropathic pain in trigeminal nerve in rat and mouse models. Low-dose PPARɣ agonist, ELB00824 (10 mg/kg), effectively decreased neuropathic hypersensitivity in mice and rats at both acute and chronic time points, a dose 100-fold lower than the effective dose (1000 mg/kg, i.p.) of pioglitazone. Comparisons of ELB00824 alone or in combination with gabapentin or carbamazepine are provided. While PPARɣ agonists used to treat Type 2 diabetes produce several adverse side effects, sub-chronic oral toxicity study provided promising results that ELB00824 does not produce any significant short-term toxicity. The study animals of either sex remained alive and healthy with no significant alteration of body weight long term. Toxicity study results obtained were satisfactory, with no significant alterations in any serum biochemistry parameters.

Sustained relief of trigeminal neuropathic pain by a blood-brain barrier penetrable PPAR gamma agonist.

Effective, non-addictive therapeutics for chronic pain remain a critical need. While there are several potential therapeutics that stimulate anti-inflammatory mechanisms to restore homeostasis in the spinal dorsal horn microenvironment, the effectiveness of drugs for neuropathic pain are still inadequate. The convergence of increasing knowledge about the multi-factorial mechanisms underlying neuropathic pain and the mechanisms of drug action from preclinical studies are providing the ability to create pharmaceuticals with better clinical effectiveness. By targeting and activating the peroxisome proliferator-activated receptor gamma subunit (PPARγ), numerous preclinical studies report pleiotropic effects of thiazolidinediones (TDZ) beyond their intended use of increasing insulin, including their anti-inflammatory, renal, cardioprotective, and oncopreventative effects. Several studies find TDZs reduce pain-related behavioral symptoms, including ongoing secondary hypersensitivity driven by central sensitization. Previous studies find increased PPARγ in the spinal cord and brain regions innervated by incoming afferent nerve endings after the induction of neuropathic pain models. PPARγ agonist treatment provides an effective reduction in pain-related behaviors, including anxiety. Data further suggest that improved brain mitochondrial bioenergetics after PPARγ agonist treatment is a key mechanism for reducing hypersensitivity. This review emphasizes two points relevant for the development of better chronic pain therapies. First, employing neuropathic pain models with chronic duration is critical since they can encompass the continuum of molecular and brain circuitry alterations arising over time when pain persists, providing greater relevance to clinical pain syndromes. Assisting in that effort are preclinical models of chronic trigeminal pain syndromes. Secondly, considering the access to nerve and brain neurons and glia across the blood-brain barrier is important. While many therapies have low brain penetrance, a PPARγ agonist with better brain penetrance, ELB00824, has been developed. Purposeful design and recent comparative testing indicate that ELB00824 is extraordinarily efficient and efficacious. ELB00824 provides greatly improved attenuation of pain-related behaviors, including mechanical hypersensitivity, anxiety, and depression in our chronic trigeminal nerve injury models. Physiochemical properties allowing significant brain access and toxicity testing are discussed.

Building and Testing PPARγ Therapeutic ELB00824 with an Improved Therapeutic Window for Neuropathic Pain.

Few models exist that can control for placebo and expectancy effects commonly observed in clinical trials measuring '' pharmacodynamics. We used the Foramen Rotundum Inflammatory Constriction Trigeminal Infraorbital Nerve injury (FRICT-ION) model to measure the effect of "full-spectrum" whole plant extracted hemp oil on chronic neuropathic pain sensitivity in mice.

The Therapeutic Effectiveness of Full Spectrum Hemp Oil Using a Chronic Neuropathic Pain Model.

In the lumbar spinal cord dorsal horn, release of afferent nerve glutamate activates the neurons that relay information about injury pain. Here, we examined the effects of protein tyrosine kinase (PTK) inhibition on NMDA receptor NR1 subunit protein expression and subcellular localization in an acute experimental arthritis model. PTK inhibitors genistein and lavendustin A reduced cellular histological translocation of NMDA NR1 in the spinal cord occurring after the inflammatory insult and the nociceptive behavioral responses to heat. The PTK inhibitors were administered into lumbar spinal cord by microdialysis, and secondary heat hyperalgesia was determined using the Hargreaves test. NMDA NR1 cellular protein expression and nuclear translocation were determined by immunocytochemical localization with light and electron microscopy, as well as with Western blot analysis utilizing both C- and N-terminal antibodies. Genistein and lavendustin A (but not inactive lavendustin B or diadzein) effectively reduced (i) pain related behavior, (ii) NMDA NR1 subunit expression increases in spinal cord, and (iii) the shift of NR1 from a cell membrane to a nuclear localization. Genistein pre-treatment reduced these events that occur  within 4 h after inflammatory insult to the knee joint with kaolin and carrageenan (k/c). Cycloheximide reduced glutamate activated upregulation of NR1 content confirming synthesis of new protein in response to the inflammatory insult. In addition to this  data, genistein or staurosporin inhibited upregulation of NMDA NR1 protein and nuclear translocation  after treatment of human neuroblastoma clonal cell cultures (SH-SY5Y) with glutamate or NMDA (4 h). These studies provide evidence that inflammatory activation of peripheral nerves initiates increase in NMDA NR1 in the spinal cord coincident with development of pain related behaviors through glutamate non-receptor, PTK dependent cascades.

Tyrosine Kinase Inhibitors Reduce NMDA NR1 Subunit Expression, Nuclear Translocation, and Behavioral Pain Measures in Experimental Arthritis.

Chronic pain often predicts the onset of psychological distress. Symptoms including anxiety and depression after pain chronification reportedly are caused by brain remodeling/recruitment of the limbic and reward/aversion circuitries. Pain is the primary precipitating factor that has caused opioid overprescribing and continued overuse of opioids leading to the current opioid epidemic. Yet experimental pain therapies often fail in clinical trials. Better understanding of underlying pathologies contributing to pain chronification is needed to address these chronic pain related issues. In the present study, a chronic neuropathic pain model persisting 10 weeks was studied. The model develops both anxiety- and pain-related behavioral measures to mimic clinical pain. The manganese-enhanced magnetic resonance imaging (MEMRI) utilized improved MRI signal contrast in brain regions with higher neuronal activity in the rodent chronic constriction trigeminal nerve injury (CCI-ION) model. T1-weighted MEMRI signal intensity was increased compared to controls in supraspinal regions of the anxiety and aversion circuitry, including anterior cingulate gyrus (ACC), amygdala, habenula, caudate, ventrolateral and dorsomedial periaqueductal gray (PAG). Despite continuing mechanical hypersensitivity, MEMRI T1 signal intensity as the neuronal activity measure, was not significantly different in thalamus and decreased in somatosensory cortex (S1BF) of CCI-ION rats compared to naïve controls. This is consistent with decreased fMRI BOLD signal intensity in thalamus and cortex of patients with longstanding trigeminal neuropathic pain reportedly associated with gray matter volume decrease in these regions. Significant increase in MEMRI T2 signal intensity in thalamus of CCI-ION animals was indication of tissue water content, cell dysfunction and/or reactive astrogliosis. Decreased T2 signal intensity in S1BF cortex of rats with CCI-ION was similar to findings of reduced T2 signals in clinical patients with chronic orofacial pain indicating prolonged astrocyte activation. These findings support use of MEMRI and chronic rodent models for preclinical studies and therapeutic trials to reveal brain sites activated only after neuropathic pain has persisted in timeframes relevant to clinical pain and to observe treatment effects not possible in short-term models which do not have evidence of anxiety-like behaviors. Potential improvement is predicted in the success rate of preclinical drug trials in future studies with this model.

Manganese-enhanced MRI reveals changes within brain anxiety and aversion circuitry in rats with chronic neuropathic pain- and anxiety-like behaviors.

An easily induced preclinical trigeminal neuropathic nerve injury model is described here for the study of chronic pain, the model acronym oramen otundum nflammatory onstriction rigeminal nfrarbital erve). In patients, neuropathic pain is thought to be related to vascular alignment or multiple sclerosis along this small trigeminal nerve branch (V2) innervating the maxillary teeth and middle third of the face. With no detectable outward physical signs, the FRICT-ION model is ideal for blinded studies. The acronym FRICT-ION applied relates to the persistence of the trigeminal neuropathic pain model likely due to sliding irritation with normal chewing in the mice. A step-by-step method to induce the mild chronic rodent neuropathic pain model is described here. The surgery is performed orally through a tiny surgical slit inside the cheek crease to align a chromic gut suture irritant along the nerve as it passes into the skull. The model allows testing of non-evoked subjective measures and evoked quantitative mechanical hypersensitivity (allodynia) testing with von Frey filaments through at least 10-14 weeks (100 days). Anxiety and depression behaviors develop within 3-6 weeks relevant to the affective component of chronic pain. While many pain drugs have failed based on testing performed in the acute animal models available, the more stable and easily replicated trigeminal inflammatory compression model is the better suited for understanding both mechanistic and affective components of nerve injury-induced chronic neuropathic pain states as well as the more ideal for preclinical trials of novel non-opioid pain relief therapies.

Minimally Invasive Oral Surgery Induction of the FRICT-ION Chronic Neuropathic Pain Model.

Acute pancreatitis (AP) and recurring AP are serious health care problems causing excruciating pain and potentially lethal outcomes due to sepsis. The validated caerulein- (CAE) induced mouse model of acute/recurring AP produces secondary persistent hypersensitivity and anxiety-like behavioral changes for study.

Effect of acetyl-L-carnitine on hypersensitivity in acute recurrent caerulein-induced pancreatitis and microglial activation along the brain's pain circuitry.

Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.

Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice.

Chemotherapy-induced neuropathic pain (CINP) is a debilitating and difficult-to-treat side effect of chemotherapeutic drugs. CINP is marked with oxidative stress and neuronal hypersensitivities. The peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that regulates genes involved in oxidative stress and inflammation. We hypothesize that PPARγ agonists are protective against CIPN by reducing oxidative stress and inhibiting neuronal hypersensitivities. To test our hypothesis, acute or chronic CIPN was introduced by short or long-term treatment of oxaliplatin in BALB/c mice. CIPN mice were treated with either a novel blood-brain barrier (BBB) penetrable PPARγ agonist ELB00824, or a BBB non-penetrable PPARγ agonist pioglitazone, or vehicle. Cold allodynia, mechanical allodynia, motor coordination, sedation and addiction were measured with dry ice, von Frey filaments, beam-walking tests, and conditioned place preference, respectively. Oxidative stress was accessed by measuring byproducts of protein oxidation (carbonyl and 3-Nitrotyrosine) and lipid peroxidation [Thiobarbituric acid reactive substances (TBARS)], as wells as gene expression of Cat, Sod2, Ppargc1a. The effects of ELB00824 on nociceptor excitability were measured using whole-cell electrophysiology of isolated dorsal root ganglion neurons. Preemptive ELB00824, but not pioglitazone, reduced oxaliplatin-induced cold and mechanical allodynia and oxidative stress. ELB0824 suppressed oxaliplatin-induced firing in IB4 neurons. ELB00824 did not cause motor discoordination or sedation/addiction or reduce the antineoplastic activity of oxaliplatin (measured with an MTS-based cell proliferation assay) in a human colon cancer cell line (HCT116) and a human oral cancer cell line (HSC-3). Our results demonstrated that ELB00824 prevents oxaliplatin-induced pain, likely via inhibiting neuronal hypersensitivities and oxidative stress.

Peroxisome proliferator-activated receptor gamma agonist ELB00824 suppresses oxaliplatin-induced pain, neuronal hypersensitivity, and oxidative stress.

A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of  = 1.794 × 10 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.

Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain.

Trigeminal neuralgia is a heterogeneous disorder with likely multifactorial and complex etiology; however, trigeminal nerve demyelination and injury is observed in almost all patients with trigeminal neuralgia. The current management strategies for trigeminal neuralgia primarily involve anticonvulsants and surgical interventions, neither of which directly address demyelination, the pathological hallmark of trigeminal neuralgia and treatments targeting demyelination are not available. Demyelination of the trigeminal nerve has been historically considered a secondary effect of vascular compression, and as a result, trigeminal neuralgia is not recognized nor treated as a primary demyelinating disorder. In this article, we review the evolution of our understanding of trigeminal neuralgia and provide evidence to propose its potential categorization, at least in some cases, as a primary demyelinating disease by discussing its course and similarities to multiple sclerosis, the most prevalent central nervous system demyelinating disorder. This proposed categorization may provide a basis in investigating novel treatment modalities beyond the current medical and surgical interventions, emphasizing the need for further research into demyelination of the trigeminal sensory pathway in trigeminal neuralgia. PERSPECTIVE: This article proposes trigeminal neuralgia as a demyelinating disease, supported by histological, clinical, and radiological evidence. Such categorization offers a plausible explanation for controversies surrounding trigeminal neuralgia. This perspective holds potential for future research and developing therapeutics targeting demyelination in the condition.

Article	Year
Sustained relief of trigeminal neuropathic pain by a blood-brain barrier penetrable PPAR gamma agonist. Molecular pain\| PubMed ID: 31588847	2019
Building and Testing PPARγ Therapeutic ELB00824 with an Improved Therapeutic Window for Neuropathic Pain. Molecules (Basel, Switzerland)\| PubMed ID: 32138198	2020
The Therapeutic Effectiveness of Full Spectrum Hemp Oil Using a Chronic Neuropathic Pain Model. Life (Basel, Switzerland)\| PubMed ID: 32443500	2020
Tyrosine Kinase Inhibitors Reduce NMDA NR1 Subunit Expression, Nuclear Translocation, and Behavioral Pain Measures in Experimental Arthritis. Frontiers in physiology\| PubMed ID: 32536874	2020
Manganese-enhanced MRI reveals changes within brain anxiety and aversion circuitry in rats with chronic neuropathic pain- and anxiety-like behaviors. NeuroImage\| PubMed ID: 32898676	2020
Minimally Invasive Oral Surgery Induction of the FRICT-ION Chronic Neuropathic Pain Model. Bio-protocol\| PubMed ID: 33659557	2020
Effect of acetyl-L-carnitine on hypersensitivity in acute recurrent caerulein-induced pancreatitis and microglial activation along the brain's pain circuitry. World journal of gastroenterology\| PubMed ID: 33727771	2021
Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice. International journal of molecular sciences\| PubMed ID: 34948407	2021
Peroxisome proliferator-activated receptor gamma agonist ELB00824 suppresses oxaliplatin-induced pain, neuronal hypersensitivity, and oxidative stress. Neuropharmacology\| PubMed ID: 36007855	2022
Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of ... International journal of molecular sciences\| PubMed ID: 37446213	2023
Trigeminal Neuralgia as A Primary Demyelinating Disease: Potential Multimodal Evidence and Remaining Controversies. The journal of pain\| PubMed ID: 37643657	2023

Karin N. Westlund

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