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Eukaryon

Trigeminal Trophic Syndrome: Analysis of TRPV1 and PRDM12 and Their Influence on Chronic Itch Disorders

Alice Glowacki, Adam Khan, Alexandra Skoczek, and Tom Steen
Department of Neuroscience and Biology
Lake Forest College
Lake Forest, Illinois 60045

Mechanosensation provides us the ability to feel the world around us. Two aspects of mechanosensation, pain and itch, are evolutionarily advantageous in keeping away harmful stimuli. However, there is a realm of disorders that come with malfunctioned pain and itch sensation. These disorders fall on a spectrum of feeling itch or pain always (atopic dermatitis and fibromyalgia), never feeling itch or pain (Congenital insensitivity to pain), and many in the middle. It is currently unknown as to why there is such a large range in the ability to sense pain and itch. Trigeminal Trophic Syndrome (TTS) lands under “I always feel itchy, but I never feel pain”, causing individuals to scratch their face off due to not being able to sense when to stop itching. This is particularly interesting because this disorder causes a possible hypersensitivity to itch and an insensitivity to pain. Two genes are specifically related to similar pathologies of itch and pain compared to TTS. TRPV1 is an established ion channel that is associated with pain and itch regulation. Dysfunction in it can lead to an unrelenting itch. PRDM12 has been found to malfunction in disorders where individuals fail to sense pain. Because each of these genes are implicated in the itch and pain mechanosensation pathways, we have hypothesized that in those with TTS, we will find malfunctions in the mechanisms of TRPV1 and PRDM12. By inducing TTS in mouse models, we hope to utilize measurements that will demonstrate aberrant levels of TRPV1 and PRDM12 as well as demonstrate confirmations that a TTS behavioral phenotype will be unveiled through modifying TPRV1 and PRDM12

Background

 

Itch (pruritus) is a very common, unpleasant sensation that triggers a desire or reflex to scratch (1).  Whereas acute itch is transient, chronic itch is a persistent, debilitating, multidimensional condition that shares many sensory similarities with pain. The vast complexity and sensory differentiation of itch necessitates appropriate classification. A proposed mechanism-based categorization distinguishes types of itch induced in a healthy nervous system by peripheral and central mechanisms(2).  Chronic neuropathic itch develops from neuronal damage of the CNS or PNS, and these disorders include multiple sclerosis, peripheral neuropathy, certain brain tumors, and nerve compression or irritation(2).   These neurogenic itch conditions result in either desensitization to pain or hypersensitization to pain accompanied by burning or stinging(1,2)

Itch is classified as a small-fiber-mediated protective (nocifensive) sensation similar to that of pain. Pain and itch are evolutionarily advantageous systems that allow organisms to avoid harmful situations(4). The pain that comes from touching a hot stove will remind us never to touch the stove again; itch is thought to have evolved to protect against small threats that would not be effectively removed with the withdrawal response associated with sensing pain. There are distinct yet complex interactions between itch and pain as evidenced by the world of disorders involving different sensitivities to itch and pain(5). Aberrant spinal processing of pain and itch result in hypersensitivity or hyposensitivity to both pain and itch(6). Chronic neuropathic itch is often associated with other significant clinical symptoms including neuropathic pain, hypersensitization, and in rare cases, hyposensitization(7,8). From a molecular level of channel dysfunction to cellular differentiation in the trigeminal ganglion(8) and dorsal root ganglion (DRG) (9) pain disorders have been shown to be related with sensory itch pathways(4,5). However, the mechanisms involved in chronic itch disorders that result in hyper- or hypo- sensitivity to pain are not well known.

The TRP channel family has been associated with pain and itch regulation(10). One focus on the pain pathway research is set on the Transient Receptor Potential Cation Channel Subfamily V Member 1 (TRPV1), which is a capsaicin receptor that is responsible for the detection of painful heat stimuli on both glabrous and non-glabrous skin(11,12). When activated by either capsaicin or heat related painful stimuli, the TRPV1 receptor opens and allows for the influx of calcium ions into the cell13). This influx of calcium into the cell can be measured and analyzed using a sensitive protein calcium sensor GCaMP6(14). When TRPV1 is active in the cell, calcium will influx, causing GCaMP6 to  label the active cell. The cellular control of the pain pathway seen in TRPV1 channels originates from the differentiation of progenitor cells into nociceptive or pruritic sensory cells.

The PRDM family is responsible for the transcription of RNA code into proteins that then determine the fate of sensory neurons(15).  PR homology domain-containing member 12 gene (PRDM12) has been associated with the differentiation of progenitor cells into sensory neurons(16). PRDM12 is a transcription factor that relies on histone modifications to then express the proper proteins that allow neurogenesis of nociceptive cells(17). Mutations in the PRDM12 gene or dysregulation of histone modification on this gene have been linked to congenital insensitivity to pain, the inability to feel pain(17). Various dysfunctions in pain and itch sensations require specific mechanisms of study to elucidate the underlyingmolecular and cellular mechanisms.

Although there are multiple genes that are implicated in pain and itch functions, they have never been studied in the same pathway. TRPV1 is shown to play a role in sensory ganglia in rodents(18). It has been suggested that PRDM12 may play a critical role in the proliferation of trigeminal ganglia of mammals(16). Lesions or injury of the trigeminal nerve can cause chronic itch syndromes, and increased or decreased sensitization to neuropathic pain. Individuals with trigeminal trophic syndrome (TTS) experience neuropathic itch as well as pain desensitization to the degree where persistent scratching behavior can lead to severe, painless self-injury(19). The phenotype of chronic itch and decreased sensitization to pain seen in trigeminal trophic syndrome represents an interaction of the itch and pain pathways. This neuropathic itch condition is  one of many conditions where itch and pain roles interact in varying degrees. As summarized in aim 1, we will be creating models for the various disorders that are on the spectrum of dysfunction. We will use aims two and three to explore the over and under expression of TRPV1 and PRDM12 in the specific TTS mouse model. Our final aim will step back to the spectrum of disorders and evaluate the over and under expression of TRPV1 and PRDM12 in these models. Nerve compressions induced by spinal cord injury (SCI) can induce chronic neurogenic itch, and hypersensitivity to both itch and pain. Conditions like atopic dermatitis and fibromyalgia are characterized by the malfunctioning one sensation,  leading to hypersensitivity to itch and pain respectively. The itch and pain mechanisms involved in neurogenic itch conditions have the potential to become treatment options that target the specific symptoms. The underlying mechanisms responsible for the aberrant sensitivities in these conditions are not known, and their interaction with one another has yet to be clearly elucidated. However, the similarities in the location and function of TRPV1 and PRDM12 suggest that they may play a role in regulating similar systems of pain and itch in TTS. We predict that modulating TRPV1 in accordance with PRDM12 will reveal the underlying dysfunction of theTTS, as well asother Itch/Pain associated disorders. We hypothesize that TRPV1 is overexpressed, and mediates itch in TTS mouse models, while PRDM12 is under -expressed in mice that feel no pain. We further predict that both TRPV1 and PRMD12 are overexpressed in the SCI and FM conditions, accounting for their hypersensitivity to pain. We also predict high TRPV1 expression paired with unaltered PRDM12 expression levels in AD.

 

Relevance

Broader Relevance:  Despite understated research, chronic itch rivals the discomfort caused by pain, and significantly impacts quality of life. Chronic itch affects approximately 15-20% of the U.S. population, and significantly impacts several quality of life parameters such as mood, concentration, eating habits, sexual function, and sleep, (20). Approximately 8% of all chronic itch conditions are neuropathic in nature, and TTS accounts for a small portion of these cases(1). Patients with TTS have an intractable neuropathic itching  accompanied with profound cutaneous denervation that makes scratching painless. Desensitization to pain paired with reflexive scratching can lead to catastrophic results. There are few effective treatment options available for TTS(2). Medications effective for conventional itching, such as antihistamines and topical steroids, fail to generate a response for neuropathic itching(1). Research advancements in elucidating how itch and pain are mediated in TTS might increase our understanding of chronic itching associated with varying pain sensitivities and possibly identify potential targets for therapy.

 

Intellectual Relevance: Chronic itch and pain desensitization in TTS is understood at a minimal level. Very little is known about the cellular and molecular bases of itching under pathological conditions where pain sensitivity is altered. Similarly, little is known about the mechanisms underlying the different conditions found on the wide scale of itch and pain sensitivity, and how these underlying mechanisms may interact to lead to a dual aberrant itch and pain sensitivity. Conducting the research laid out in this proposal at the cellular and molecular levels could expand our understanding of the roles of pain-associated and itch-implicated TRPV1 ion channel and the pain-associated gene PRDM12 in TTS. Our proposed approaches may also expand our knowledge in studying chronic neurogenic itch and/or pain conditions. Success in elucidating the role and possible interaction of TRPV1 and PRDM12 could provide insight on TTS and other conditions for future researchers and provide possible molecular targets for treatment options.

 

Specific Aims

The aim of our proposal is to evaluate the role of TRPV1 and PRDM12 in mouse models of TTS and other chronic itch and/or pain conditions by genetically and chemically manipulating the expression and function of TRPV1 and PRDM12 after first determining the baseline or natural levels of TRPV1 and PRDM12 in these chronic itch/pain conditions.

 

  1. Profile the expression and functionality of TRPV1 and PRDM12 in multiple chronic itch/pain conditions. Mice exhibiting TTS, SCI, atopic dermatitis, and fibromyalgia will be generated following established protocols. The expression and functionality of TRPV1 and PRDM12 will be assessed in each mouse model.
  2. Analyze the effects of genetically or chemically over expressed or under expressed TRPV1 in TTS. In the mouse model for trigeminal trophic syndrome TRPV1 will be both over expressed and under expressed using both genetic and chemical methods.  Two different physiological imaging techniques along with two different behavioral analyses will be done to examine the difference of TRPV1 when compared to the WT disease model.  TRPA and ASIC will be used as controls.
  3. Analyze the effects of genetic modifications to upregulate or downregulate PRDM12 in TTS. Following the experiments done on TRPV1, PRDM12 will be upregulated and downregulated in a similar mouse model for TTS. We will then measure the effects of each regulation through various methods that will measure developmental impact. Kdm4a and H3K9me will be used as controls for measuring the normal expression of PRDM12
  4. Analyze the effect of genetically or chemically over expressed or under expressed TRPV1 and upregulated or downregulated PRDM12 in AD, SCI and FM mouse models. Experiments on TRPV1 and PRDM12 within TTS mouse models will be replicated in the other three itch/pain conditions (SCI, AD, FM).

 

Research Methods and Design

 

  1. Profile expression & functionality of TRPV1 & PRDM12 in multiple chronic itch/pain conditions.

Rationale: Neurogenic itch conditions result in desensitization or hypersensitization to pain that is often accompanied by burning or stinging(2). Pain and itch sensitivity can be measured on a scale ranging from hypo- to hyper- sensitivity. We plan to generate mouse models exhibiting different disorders that fall on different places on this sensitivity scale, including TTS (hypersensitivity to itch; hyposensitivity to pain), atopic dermatitis (hypersensitivity to itch; normal sensitivity to pain), SCI (hypersensitivity to both itch and pain), and fibromyalgia (normal sensitivity to itch; hypersensitivity to pain). We plan to analyze the expression and functionality of TRPV1 and PRDM12 in these conditions in order to assess their roles in mediating itch and pain. Analysis of TRPV1 expression will include RT-PCR, Western Blot, and immunohistochemistry, while functionality will be analyzed with calcium imaging techniques. Analysis of PRDM12 expression will include in situ hybridization, RT-PCR, Western Blot, and immunofluorescence, while functionality will be analyzed with chromatin immunoprecipitation. Results will be analyzed using ANNOVA and paired-t tests.

A1. Generation of Trigeminal Trophic Syndrome mouse model (chronic itch & pain desensitization): We will induce TTS in mice by partially ligating the infraorbital (ION) branch of the trigeminal nerve on the left side of mice(21). Our controls will include sham mice and normal control mice. For sham-operated mice, the ION will be exposed on the right side using the same procedure, but will not be ligated(21).

A2. Generation of Spinal Cord Injury induced neurogenic itch model (chronic itch & pain sensitization): We will induce SCI in mice with unilateral intraspinal injections of the AMPA metabotropic receptor agonist quisqualic acid (QUIS) at the dorsal horn and intermediate gray (right side) in spinal segments of mice ranging from T12 to L2(22). Our controls will include WT mice that receive no injections, and sham mice that will receive an intraspinal injection of a vehicle compound.

A3. Generation of Atopic Dermatitis mouse model (chronic itch and no pain):  We will induce atopic dermatitis by intraperitoneally inoculating mice with ovalbumin (OVA). Controls will include WT mice with no inoculation, and sham mice that will receive aluminum hydroxide(19).

A4. Generation of fibromyalgia mouse model (chronic pain and no itch):  We will use the biogenic amine depletion mouse model to study fibromyalgia in mice. Repeated subcutaneous administration of reserpine will induce hyperalgesia by significantly decreasing the amount of biogenic amines in the spinal cord, thalamus, and prefrontal cortex(24).  Our controls will include sham mice, which will receive vehicle injections in the same subcutaneous locations, and normal control mice.

B1. Analysis of TRPV1 expression:  The natural expression and functionality of TRPV1 will be assessed in each mouse model previously generated in this aim. Mice will be sacrificed at 1, 3, 7, 14, and 60 days after each pathological condition induction(21). The spinal cord, frontal cortical region, and DRG will then be removed, and TRPV1 and TRPA1 mRNA levels will be quantified by RT- PCR, while their protein levels will be assessed with Western Blot. These regions will also be processed for immuno-histochemical staining as described by Kim et al. 2008 using guinea pig anti-mouse TRPV1 and FITC-conjugated donkey anti-rabbit IgG (red)(24). Activating rabbit anti-human activated transcription factor-3 (ATF3) will be used as a neuronal injury marker to distinguish TG neurons into either injured or uninjured neurons and then compared to the expression patterns of TRPV1 between injured and uninjured TG neurons(21).

B2. Analysis of TRPV1 functionality:  The functionality of TRPV1, ASIC, and TRPA1 will be assessed by performing calcium imaging experiments using Fura-2 as the indicator as described by previous studies(22). Patch-clamp recordings will assess calcium free expression in the trigeminal ganglia. Images will be done using genetically encoded calcium indicators (GECIs), specifically GCamP(27).

C1. Analysis of PRDM12 expression:  The natural expression and functionality of PRDM12 will be assessed in each mouse model previously generated in this aim. PRDM12 protein levels in the spinal cord, DRG, neural crest, neural fold, and trigeminal placode neuron will be assessed using Western Blot, while PRDM12 mRNA levels will be quantified using RT-PCR. Since previous studies have shown that PRDM12 expression is most prominent during embryonic and early development, we will use in situ hybridization on whole-mount and whole-embryos to assess PRDM12 expression throughout embryonic mice development(17). Immunofluorescence/immunohistochemistry will be performed throughout embryonic development and at 1, 3, 7, 14, and 60 days’ post condition induction using the PRDM12 pCMV6 vector with a DDK-tag at C-terminus, which will then be washed with Alexa Fluor 568 goat anti-mouse antibodies(16). Immunofluorescence will be used to compare WT and condition models.

C2. Analysis of PRDM12 functionality: We will use chromatin immunoprecipitation-sequencing (ChIP-Seq) experiments to assess PRDM12 functionality throughout these condition models using mouse Flag-Prdm12 and anti-Flag antibodies(28, 29).

 

Prediction: As a neurogenic itch condition accompanied by pain desensitization, we expect TTS mice to have significantly decreased TRPV1 and PRDM12 expression levels and functionality. Since SCI-induced neurogenic chronic itch conditions and fibromyalgia are accompanied with hypersensitization to pain, we expect TRPV1 and PRDM12 expression and functionality levels to be significantly elevated in comparison to controls for both conditions. As atopic dermatitis is a chronic itch condition with no association with pain, we expect elevated TRPV1 expression and functionality levels in comparison to controls, while expression and functionality of PRDM12 levels should mirror controls.

 

  1. Analyze the effect of genetically or chemically over expressed or under expressed TRPV1 in TTS.

Rationale:  TRPV1 is a capsaicin receptor that is responsible for the detection of painful heat stimuli on both glabrous and non-glabrous skin(12).  We plan to use genetically mutated mouse models to both overexpress and under-express TRPV1.  Pharmacological methods will then be used for the same purpose.  Similar genetic and chemical processes will be used to overexpress and under-express TRPA and ASIC, which will be used as the controls due to both being other channels implicated in pain sensation.  Calcium imaging techniques as well as immunohistochemical staining will then be used to analyze the cellular activity of TRPV1, ASIC, and TRPA.  Behavioral analysis will be done to look at WT disease models and TRPV1 over and under expression.  Studying the effects of TRPV1 overexpression and under expression compared to WT diseased mice will provide evidence that TRPV1 channels are implicated in the altered pain sensation characteristic of  the chronic itch disorder, trigeminal trophic syndrome.

 

A1. Generation of genetically overexpressed TRPV1 mouse models:  In order to overexpress TRPV1 in the WT disease model, Cre-loxP conditional expression will be used.  A homologous recombination will be inserted in the ROSA26 locus in order to facilitate increased expression by Cre recombinase(30).  Cre-loxP will also be used in order to conditionally overexpress ASIC and TRPA, both of which will be used as controls.  Once overexpression is done, the trigeminal nerve will be severed to induce TTS.

A2.  Generation of genetically under expressed TRPV1 mouse models:  In order to knock-out TRPV1 in the mice, the gene coding for TRPV1, VR1, will be manipulated(9).  The exon that encodes for the 5th and 6th transmembrane domains and the pore-loop region will be deleted.  The genetic manipulation has been previously shown to induce TRPV1-/- animals(31).   Similar to TRPV1, ASIC and TRPA will also be knocked out and used as controls.  After under expression is done, the trigeminal nerve will again be severed to induce TTS.

B1.  Generation of pharmacologically induced overexpressed TRPV1: For chemical overexpression of TRPV1, capsaicin will be injected in a low dose in order to increase expression(32).  ASIC and TRPA will be used as controls.  Chemical overexpression of ASIC will be done using injection of extracellular protons, specifically injection of Li+(33).  Chemical overexpression of TRPA will be done by using injections of allicin(30).  Injections will be done after severing the trigeminal nerve and inducing TTS.

B2.  Generation of pharmacologically induced under expressed TRPV1: TRPV1 will be pharmacologically under expressed by ablation using RTX.  RTX will be administered intrathecally and testing will be done 24 hours after administration(31).  Amiloride will be used in order to pharmacologically inhibit ASIC(36).  To pharmacologically inhibit TRPA, A-967079, an antagonist of TRPA, will be used(33).

C1.  Calcium imaging techniques:  In order to analyze the expression of TRPV1, ASIC, and TRPA in both the WT diseased model as well as the genetic and chemically altered models, calcium imaging will be done.  Images will be done using genetically encoded calcium indicators (GECIs), specifically GCamP(38).

C2. Immunohistochemical staining: Immunofluorescence will be performed using the TRPV1 monoclonal antibody 10E3-1A2, the rabbit anti-rat calcitonin gene-related peptide (CGRP) polyclonal antiserum and immune complexes will be stained with Alexa Fluor 568 goat anti-rabbit or goat anti-mouse secondary antibody(26).  Immunofluorescence will be used to compare both WT disease and genetically and chemically altered animals.  Immunofluorescence will also be used to analyze over and under-expression of TRPA and ASIC.

D1. Behavioral analysis using Bradykinin:  In order to determine the TRPV1 differences when mice are both chemically and genetically overexpressed and under-expressed, Bradykinin will be injected into the plantar surface of a hind paw(39).  Bradykinin will induce a brief, painful stimulus when injected(40).  The licking response of the animals will be measured to analyze the level of pain both the WT and TRPV1 altered animals experience.

D2. Behavioral analysis using histamine:  In order to analyze the TRPV1 differences when mice are both chemically and genetically overexpressed and under-expressed, histamine will be used to induce itch behavior.  Histamine will be given at a concentration of 3-300 μg.  The  amount of time the animals spend itching and biting will then be analyzed to determine the amount of itch sensation that they felt(41).

 

Prediction:  The knock-in of TRPV1 in disease mouse models will lead to more TRPV1 activation when viewed with calcium imaging techniques as well as immunohistochemical staining.  Knockout of TRPV1 in disease mouse models will lead to less activation of TRPV1 when imaged.  Similar to genetic manipulations, chemically induced over expression will lead to more TRPV1 activation, and chemically induced under-expression will lead to less TRPV1 activation. The controls of TRPA and ASIC will show little to no effect with over and under expressed when compared to TRPV1.  Behavioral analysis of overexpression and under-expression of TRPV1 compared to WT disease models will show that when TRPV1 is overexpressed the animals will have more pain and itch sensation behaviors.

 

  1. Analyze the effect of genetic modification to upregulate or downregulate PRDM12 in TTS.

Rationale: Because we have predicted that TRPV1 overexpression will reflect the itch phenotype of TTS, the PRDM12 transcription factor is what controls for the differentiation of progenitor cells into pain sensing neurons(16). An increase in PRDM12 expression during days eight and nine of neuronal development in mouse models appears to be a mechanism in which sensory neurons develop into pain cells. Loss of function mutations have shown a significant decrease in the amount of PRDM12 expression and associated pain sensing neurons(17). As a control, we will analyze the putative effects of PRMD12 in the previously established TTS mouse model from our first aim. After performing a knockdown of PRDM12, we will attempt to restore its function at the transcriptional level of control. Established behavioral pain perception tests will be used in each model to assess the effects of neuronal modification on the itch and pain phenotype.

A1. Generation of genetically overexpressed PRDM12 embryo mouse models: In the same line of thought as overexpressing TRPV1, Cre-loxP will be used for a knock-in mouse model to overexpress PRDM12.  A homologous recombination will be inserted in the ROSA26 locus in order to facilitate increased expression by Cre recombinase(30). We will use a Wild-type mouse as a control. The PRDM12 knock-in will be done on a Wild-type mouse to serve as a control and ensure that our knock in-method works. We will then perform the knock-in on the previously established TTS mouse. This will serve as our measure to test if increasing expression of PRDM12 will increase the itch phenotype. Western Blot analyses will be done in each experimental condition to ensure that our manipulations work. The TTS mouse model will be manipulated in each of the aims and we will use a WT and mutant mouse as controls.

A2.  Generation of genetically under expressed PRDM12 embryo mouse models: Myc-tagged mouse PRDM12 cDNA will be used to insert PRDM12morpholino inside expression vector pCMV-myc(28). This will serve as the knockdown model of PRDM12 to measure the effects of its absence. This knockdown will be done to the TTS mouse to study the effects of decreasing PRDM12 levels in a disease model. We will compare the knock down to a wild-type mouse, as well as the TTS mouse model.

B1. Generation of pharmacologically induced overexpressed PRDM12: Since PRDM12 has been shown to be induced by retinoic acid, we will apply retinoic acid receptor beta 2 (RARβ2) agonist CD2019(45,,46). This agonist will be used to increase the expression of PRDM12 associated neurite outgrowth, through increasing the functionality and expression of RARβ2.

B2. Generation of pharmacologically induced under expressed PRDM12: To decrease the amount of PRDM12, we will inject RARβ2 antagonist LE135, which has been shown to decrease the functionality and expression of RARβ2(43). In doing so, the inductive effects of retinoic acid on PRDM12 should be eliminated and PRDM12 will become under-expressed.

C1. Sural Nerve Biopsy to measure the amount of end stage neuronal development: Toluidine blue-stained nerve biopsies of the mutant mice will be obtained and measured for morphometric evaluation(17). This biopsy will be a quantitative measure of myelin sheaths that are present in the intact nerve fibers runningthrough each section. WT mice will be used as a control to measure the amount of normal sensory neuron expression.

C2. Immunohistochemical staining: Similar to TRPV1 Immunofluorescence will be performed using the PRDM12 pCMV6 vector with a DDK-tag at C-terminus which will then be washed with Alexa Fluor 568 goat anti-mouse antibodies(16).  Immunofluorescence will be used to compare both WT disease and genetically and chemically altered animals.  Immunofluorescence will also be used to analyze over and under expression of WT and PRDM12 mutations as controls. These will all be done throughout the development of the mice, since it has been shown that maximum PRDM12 expression occurs around day eight of development (17).

D1. Behavioral analysis using Bradykinin:  In order to determine the PRDM12 differences when mice are both chemically and genetically overexpressed and under-expressed, Bradykinin will be injected into the plantar surface of a hind paw(39).  Bradykinin will induce a brief, painful stimulus when injected(40).  The licking response of the animals will be measured to analyze the level of pain both the WT and PRDM12 altered animals experience.

D2. Behavioral analysis using histamine:  In order to analyze the PRDM12 differences when mice are both chemically and genetically overexpressed and under-expressed, histamine will be used to induce itch behavior.  Histamine will be given at a concentration of 3-300 μg.  The amount of time the animals spent itching and biting will then be analyzed to determine the amount of itch sensation that they felt(41).

 

Prediction: Overexpression of PRDM12 will lead to an increase of pain sensation in the mouse models that will become evident in mouse response to noxious stimuli. Since the TTS model mouse should already have a decrease in PRDM12 due to its phenotype of not feeling pain, overexpression of PRDM12 will attenuate itch behaviors because the mouse will now feel the pain of excessive itching. Under expression of PRDM12 will lead to a dysfunction in growth of the mouse embryos. In the mouse model, under expression will lead to an exacerbation of the itch phenotype. A sural nerve biopsy should demonstrate a decrease in overall sensory neuron presence in the mice with over or under-expressed PRDM12. Immunohistochemical staining should reveal that genetically under expressed PRDM12 mice will have less of the PRDM12.  The PRDM12 upregulated mice will demonstrate clear phenotypes of decreased sensitivity to pain and increased itch sensation that resemble trigeminal trophic syndrome.

 

  1. Analyze the effect of genetically or chemically over expressed or under expressed TRPV1 and upregulated or downregulated PRDM12 in AD, SCI and FM mouse models.

Rationale: Aim 1 tested four different itch/pain models (TTS, SCI, AD, FM). Aims 2 & 3 focused on one model (TTS) with one manipulation (TRPV1 or PRDM12). In Aim 4 we sought to test the three different models of Aim 1 (SCI, AD, FM) in the same manner of manipulation as TTS in Aims 2 & 3.

A1. TRPV1 manipulated SCI induced mice models:

Wild type SCI induced mice(21) will serve as the control group. Both TRPV1 over-expressed and TRPV1 under-expressed SCI induced mice shall serve as the experimental groups. Two experimental conditions of bradykinin(40)  and histamine(41)  injections shall be given as well as control, saline injections. This methodology calls for nine groups of mice, with each of the three groups (WT, TRPV1 over-expressed, TRPV1 under-expressed) having three conditions (bradykinin, histamine, saline). With all injection conditions: licking response—indicating level of pain experience(36) and time spent itching & biting—indicating itch response(37) shall be measured and compared across groups.

A2. TRPV1 manipulated atopic dermatitis mice models:

Wild type AD mice(23) will serve as the control group. Both TRPV1 over-expressed and TRPV1 under-expressed AD mice shall serve as the experimental groups. Two experimental conditions of bradykinin(40)  and histamine(41)  injections shall be given as well as control, saline injections. This methodology calls for nine groups of mice, with each of the three groups (WT, TRPV1 over-expressed, TRPV1 under-expressed) having three conditions (bradykinin, histamine, saline). With all injection conditions licking response—indicating level of pain experience(40)  and time spent itching & biting—indicating itch response(40)  shall be measured and compared across groups.

A3. TRPV1 manipulated fibromyalgia mice models:

Wild type FM mice(20) will serve as the control group. Both TRPV1 over-expressed and TRPV1 under-expressed FM mice shall serve as the experimental groups. Two experimental conditions of bradykinin(40)  and histamine(41) injections shall be given as well as control, saline injections. This methodology calls for nine groups of mice, with each of the three groups (WT, TRPV1 over-expressed, TRPV1 under-expressed) having three conditions (bradykinin, histamine, saline). With all injection conditions licking response—indicating level of pain experience(40)  and time spent itching & biting—indicating itch response(41)  shall be measured and compared across groups.

B1. PRDM12 manipulated SCI induced mice models:

Wild type SCI induced mice(20) will serve as the control group. Both PRDM12 over-expressed and PRDM12 under-expressed SCI induced mice shall serve as the experimental groups. Two experimental conditions of bradykinin(40)  and histamine(40)   injections shall be given as well as control, saline injections. This methodology calls for nine groups of mice, with each of the three groups (WT, PRDM12 over-expressed, PRDM12 under-expressed) having three conditions (bradykinin, histamine, saline). With all injection conditions licking response—indicating level of pain experience(36) and time spent itching & biting—indicating itch response(37) shall be measured and compared across groups.

B2. PRDM12 manipulated atopic dermatitis mice models:

Wild type AD mice(20) will serve as the control group. Both PRDM12 over-expressed and PRDM12 under-expressed AD mice shall serve as the experimental groups. Two experimental conditions of bradykinin(40)   and histamine(41) injections shall be given as well as control, saline injections. This methodology calls for nine groups of mice, with each of the three groups (WT, PRDM12 over-expressed, PRDM12 under-expressed) having three conditions (bradykinin, histamine, saline). With all injection conditions licking response—indicating level of pain experience(40)  and time spent itching & biting—indicating itch response(41) shall be measured and compared across groups.

B3. PRDM12 manipulated fibromyalgia mice models:

Wild type FM mice(20) will serve as the control group. Both PRDM12 over-expressed and PRDM12 under-expressed FM mice shall serve as the experimental groups. Two experimental conditions of bradykinin(40)  and histamine(41) injections shall be given as well as control, saline injections. This methodology calls for nine groups of mice, with each of the three groups (WT, PRDM12 over-expressed, PRDM12 under-expressed) having three conditions (bradykinin, histamine, saline). With all injection conditions licking response—indicating level of pain experience(40)  and time spent itching & biting—indicating itch response(41) shall be measured and compared across groups.

 

Prediction: SCI TRPV1 over-expressed bradykinin injected mice will have significantly higher licking responses as compared to saline and control conditions—indicating higher levels of pain. SCI TRPV1 over-expressed histamine injected mice will spend significantly more time itching and biting, as compared to saline and control conditions—indicating greater itch sensation. AD TRPV1 over-expressed histamine injected mice will spend significantly more time itching and biting, as compared to saline and control conditions—indicating greater itch sensation. FM TRPV1 over-expressed bradykinin injected mice will have significantly higher licking responses as compared to saline and control conditions—indicating higher levels of pain. Across all three groups (SCI, AD, FM) PRDM12 over-expressed mice will exhibit both higher licking responses and more time spent itching and biting—indicating greater overall sensitivity to pain and itch—when compared to WT controls in all three injection conditions. We further predict that across all three groups (SCI, AD, FM) PRDM12 under-expressed mice will exhibit both less licking responses and less time spent itching and biting—indicating sensation inhibition to pain and itch—when compared to PRDM12 over-expressed conditions as well as WT control conditions, in all three injection conditions.

 

References

 

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