Vivianne Tawfik

All Publications

• Cell-specific overexpression of COMT in dopaminergic neurons of Parkinson's disease. Brain : a journal of neurology Kuzumaki, N., Suda, Y., Iwasawa, C., Narita, M., Sone, T., Watanabe, M., Maekawa, A., Matsumoto, T., Akamatsu, W., Igarashi, K., Tamura, H., Takeshima, H., Tawfik, V. L., Ushijima, T., Hattori, N., Okano, H., Narita, M. 2019; 142 (6): 1675–89

  Abstract

  The mechanism by which dopaminergic neurons are selectively affected in Parkinson's disease is not fully understood. In this study, we found a dramatic increase in the expression of catechol-O-methyltransferase (COMT), along with a lower level of DNA methylation, in induced pluripotent stem cell-derived dopaminergic neurons from patients with parkin (PARK2) gene mutations compared to those from healthy controls. In addition, a significant increase in the expression of COMT was found in dopaminergic neurons of isogenic PARK2 induced pluripotent stem cell lines that mimicked loss of function of PARK2 by CRISPR Cas9 technology. In dopamine transporter (DAT)-Cre mice, overexpression of COMT, specifically in dopaminergic neurons of the substantia nigra, produced cataleptic behaviours associated with impaired motor coordination. These findings suggest that upregulation of COMT, likely resulting from DNA hypomethylation, in dopaminergic neurons may contribute to the initial stage of neuronal dysfunction in Parkinson's disease.

  View details for DOI 10.1093/brain/awz084

  View details for PubMedID 31135049

• 18F-FDG PET/MRI of patients with chronic pain alters management. Cipriano, P., Yoon, D., Carroll, I., Curtin, C., Tawfik, V., Xu, Y., Biswal, S. SOC NUCLEAR MEDICINE INC. 2019

  View details for Web of Science ID 000473116800093

• Musculoskeletal changes on [18F]FDG PET/MRI from complex regional pain syndrome in foot Yoon, D., Xu, Y., Cipriano, P., Tawfik, V., Curtin, C., Carroll, I., Biswal, S. SOC NUCLEAR MEDICINE INC. 2019

  View details for Web of Science ID 000473116800094

• Microglial Modulation as a Target for Chronic Pain: From the Bench to the Bedside and Back. Anesthesia and analgesia Haight, E. S., Forman, T. E., Cordonnier, S. A., James, M. L., Tawfik, V. L. 2019; 128 (4): 737–46

  Abstract

  With a widespread opioid epidemic and profound biopsychosocial implications, chronic pain is a multifaceted public health issue requiring urgent attention. The treatment of chronic pain is particularly important to anesthesiologists given our unique role as perioperative physicians and pain medicine specialists. The present review details the recent shift from a neuronal theory of chronic pain to one that includes complex neuron-glia interactions. In particular, we highlight microglia, the myeloid-lineage cells of the central nervous system, as initiators of a postinjury neuroimmune response that contributes to the acute to chronic pain transition. We discuss ever-advancing preclinical studies, wherein significant success has been made through pharmacologic and genetic modulation of microglia, and we emphasize where these approaches have made the transition to the clinical realm. Furthermore, we highlight the most current, novel efforts to visualize glial activation in vivo using positron emission tomography and improve the diagnosis of chronic pain through radiotracer binding of specific targets, like the 18 kDa translocator protein in microglia and myeloid-lineage cells. Our rapidly advancing knowledge about microglia and their involvement in pain suggests that the era of glial-targeted therapeutics is just beginning so long as we refocus our attention on optimizing preclinical studies using a clinically informed approach, before translation.

  View details for PubMedID 30883419

• Morphine Exacerbates Postfracture Nociceptive Sensitization, Functional Impairment, and Microglial Activation in Mice ANESTHESIOLOGY Li, W., Irvine, K., Sahbaie, P., Guo, T., Shi, X., Tawfik, V. L., Kingery, W. S., Clark, J. 2019; 130 (2): 292–308

  View details for DOI 10.1097/ALN.0000000000002495

  View details for Web of Science ID 000466747200016

• Longitudinal TSPO-PET imaging of peripheral and central myeloid cells in a mouse model of complex regional pain syndrome. Pain Cropper, H. C., Johnson, E. M., Haight, E., Cordonnier, S. A., Chaney, A. M., Forman, T. E., Biswal, A., Stevens, M. Y., James, M. L., Tawfik, V. L. 2019

  Abstract

  Complex regional pain syndrome (CRPS) is a severely disabling disease characterized by pain, temperature changes, motor dysfunction and edema that most often occurs as an atypical response to a minor surgery or fracture. Inflammation involving activation and recruitment of innate immune cells, including both peripheral and central myeloid cells (i.e. macrophages and microglia, respectively), is a key feature of CRPS. However, the exact role and time-course of these cellular processes relative to the known acute and chronic phases of the disease are not fully understood. Positron emission tomography (PET) of translocator protein-18kDa (TSPO) is a method for non-invasively tracking these activated innate immune cells. Here, we reveal the temporal dynamics of peripheral and central inflammatory responses over 20 weeks in a tibial fracture/casting mouse model of CRPS through longitudinal TSPO-PET using [F]GE-180. PET tracer uptake quantification in the tibia revealed increased peripheral inflammation as early as 2 days post-fracture and lasting 7 weeks. Centralized inflammation was detected in the spinal cord and brain of fractured mice at 7 and 21 days post-injury. Spinal cord tissue immunofluorescent staining revealed TSPO expression in microglia (CD11b+) at 7 days, but was restricted mainly to endothelial cells (PECAM1+) at baseline and 7 weeks. Our data suggest early and persistent peripheral myeloid cell activation, and transient central microglial activation are limited to the acute phase of CRPS. Moreover, we show that TSPO-PET can be used to noninvasively monitor the spatiotemporal dynamics of myeloid cell activation in CRPS progression with potential to inform disease phase-specific therapeutics.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

  View details for PubMedID 31095093

• Morphine Exacerbates Postfracture Nociceptive Sensitization, Functional Impairment, and Microglial Activation in Mice. Anesthesiology Li, W., Irvine, K., Sahbaie, P., Guo, T., Shi, X., Tawfik, V. L., Kingery, W. S., Clark, J. D. 2018

  Abstract

  WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Emerging evidence suggests that opioid use immediately after surgery and trauma may worsen outcomes. In these studies, the authors aimed to determine whether morphine administered for a clinically relevant time period (7 days) in a tibia fracture orthopedic surgery model had adverse effects on postoperative recovery.METHODS: Mice were given morphine twice daily for 7 days after unilateral tibial fracture and intramedullary pin fixation to model orthopedic surgery and limb trauma. Mechanical allodynia, limb-specific weight bearing, gait changes, memory, and anxiety were measured after injury. In addition, spinal cord gene expression changes as well as glial activation were measured. Finally, the authors assessed the effects of a selective Toll-like receptor 4 antagonist, TAK-242, on nociceptive and functional changes after injury.RESULTS: Tibial fracture caused several weeks of mechanical nociceptive sensitization (F(1, 216) = 573.38, P < 0.001, fracture + vehicle vs. sham + vehicle, n = 10 per group), and this change was exacerbated by the perioperative administration of morphine (F(1, 216) = 71.61, P < 0.001, fracture + morphine vs. fracture + vehicle, n = 10 per group). In additional testing, injured limb weight bearing, gait, and object location memory were worse in morphine-treated fracture mice than in untreated fracture mice. Postfracture expression levels of several genes previously associated with opioid-induced hyperalgesia, including brain-derived neurotrophic factor and prodynorphin, were unchanged, but neuroinflammation involving Toll-like receptor 4 receptor-expressing microglia was observed (6.8 ± 1.5 [mean ± SD] cells per high-power field for fracture + vehicle vs. 12 ± 2.8 fracture + morphine, P < 0.001, n = 8 per /group). Treatment with a Toll-like receptor 4 antagonist TAK242 improved nociceptive sensitization for about 2 weeks in morphine-treated fracture mice (F(1, 198) = 73.36, P < 0.001, fracture + morphine + TAK242 vs. fracture + morphine, n = 10 per group).CONCLUSIONS: Morphine treatment beginning at the time of injury impairs nociceptive recovery and other outcomes. Measures preventing glial activation through Toll-like receptor 4 signaling may reduce the adverse consequences of postoperative opioid administration.

  View details for PubMedID 30418215

• [EXPRESS] Autoinflammatory and Autoimmune Contributions to Complex Regional Pain Syndrome. Molecular pain Clark, J. D., Tawfik, V., Tajerian, M., Kingery, W. 2018: 1744806918799127

  Abstract

  Complex regional pain syndrome (CRPS) is a highly enigmatic syndrome typically developing after injury or surgery to a limb. Severe pain and disability are common amongst those with chronic forms of this condition. Accumulating evidence suggests that CRPS may involve both autoinflammatory and autoimmune components. In this review article evidence for dysfunction of both the innate and adaptive immune systems in CPRS is presented. Findings from human studies in which cytokines and other inflammatory mediators were measured in the skin of affected limbs is discussed. Additional results from studies of mediator levels in animal models are evaluated in this context. Similarly, the evidence from human, animal and translational studies of the production of autoantibodies and the potential targets of those antibodies is reviewed. Compelling evidence of autoinflammation in skin and muscle of the affected limb has been collected from CRPS patients and laboratory animals. Cytokines including IL-1beta, IL-6, TNFalpha and others are reliably identified during the acute phases of the syndrome. More recently, autoimmune contributions have been suggested by the discovery of self-directed pain-promoting IgG and IgM antibodies in CRPS patients and model animals. Both the autoimmune and autoinflammatory components of CRPS appear to be regulated by neuropeptide containing peripheral nerve fibers and the sympathetic nervous system. While CRPS displays a complex neuroimmunological pathogenesis, therapeutic interventions could be designed targeting autoinflammation, autoimmunity or the neural support for these phenomena.

  View details for PubMedID 30124090

• Functional Divergence of Delta and Mu Opioid Receptor Organization in CNS Pain Circuits NEURON Wang, D., Tawfik, V. L., Corder, G., Low, S. A., Francois, A., Basbaum, A. I., Scherrer, G. 2018; 98 (1): 90-+

  Abstract

  Cellular interactions between delta and mu opioid receptors (DORs and MORs), including heteromerization, are thought to regulate opioid analgesia. However, the identity of the nociceptive neurons in which such interactions could occur in vivo remains elusive. Here we show that DOR-MOR co-expression is limited to small populations of excitatory interneurons and projection neurons in the spinal cord dorsal horn and unexpectedly predominates in ventral horn motor circuits. Similarly, DOR-MOR co-expression is rare in parabrachial, amygdalar, and cortical brain regions processing nociceptive information. We further demonstrate that in the discrete DOR-MOR co-expressing nociceptive neurons, the two receptors internalize and function independently. Finally, conditional knockout experiments revealed that DORs selectively regulate mechanical pain by controlling the excitability of somatostatin-positive dorsal horn interneurons. Collectively, our results illuminate the functional organization of DORs and MORs in CNS pain circuits and reappraise the importance of DOR-MOR cellular interactions for developing novel opioid analgesics.

  View details for PubMedID 29576387

  View details for PubMedCentralID PMC5896237

• TEMPORAL CONTROL OF MICROGLIAL REACTIVITY REVEALS SEX-INDEPENDENT FUNCTIONAL CONTRIBUTION OF MICROGLIA TO LONG-LASTING PAIN Takemura, Y., Clark, D., Forman, T., Haight, E., Tawfik, V. LIPPINCOTT WILLIAMS & WILKINS. 2018: 437

  View details for Web of Science ID 000460106500249

• Activation of ventral tegmental area dopaminergic neurons reverses pathological allodynia resulting from nerve injury or bone cancer MOLECULAR PAIN Watanabe, M., Narita, M., Hamada, Y., Yamashita, A., Tamura, H., Ikegami, D., Kondo, T., Shinzato, T., Shimizu, T., Fukuchi, Y., Muto, A., Okano, H., Yamanaka, A., Tawfik, V. L., Kuzumaki, N., Navratilova, E., Porreca, F., Narita, M. 2018; 14: 1744806918756406

  Abstract

  Chronic pain induced by nerve damage due to trauma or invasion of cancer to the bone elicits severe ongoing pain as well as hyperalgesia and allodynia likely reflecting adaptive changes within central circuits that amplify nociceptive signals. The present study explored the possible contribution of the mesolimbic dopaminergic circuit in promoting allodynia related to neuropathic and cancer pain. Mice with ligation of the sciatic nerve or treated with intrafemoral osteosarcoma cells showed allodynia to a thermal stimulus applied to the paw on the injured side. Patch clamp electrophysiology revealed that the intrinsic neuronal excitability of ventral tegmental area (VTA) dopamine neurons projecting to the nucleus accumbens (N.Acc.) was significantly reduced in those mice. We used tyrosine hydroxylase (TH)-cre mice that were microinjected with adeno-associated virus (AAV) to express channelrhodopsin-2 (ChR2) to allow optogenetic stimulation of VTA dopaminergic neurons in the VTA or in their N.Acc. terminals. Optogenetic activation of these cells produced a significant but transient anti-allodynic effect in nerve injured or tumor-bearing mice without increasing response thresholds to thermal stimulation in sham-operated animals. Suppressed activity of mesolimbic dopaminergic neurons is likely to contribute to decreased inhibition of N.Acc. output neurons and to neuropathic or cancer pain-induced allodynia suggesting strategies for modulation of pathological pain states.

  View details for PubMedID 29357732

• Successful treatment of chronic knee pain following localization by a sigma-1 receptor radioligand and PET/MRI: a case report JOURNAL OF PAIN RESEARCH Cipriano, P., Lee, S., Yoon, D., Shen, B., Tawfik, V., Curtin, C., Dragoo, J. L., James, M., Mccurdy, C., Chin, F., Biswal, S. 2018; 11: 2353–56

  Abstract

  The ability to accurately diagnose and objectively localize pain generators in chronic pain sufferers remains a major clinical challenge since assessment relies on subjective patient complaints and relatively non-specific diagnostic tools. Developments in clinical molecular imaging, including advances in imaging technology and radiotracer design, have afforded the opportunity to identify tissues involved in pain generation based on their pro-nociceptive condition. The sigma-1 receptor (S1R) is a pro-nociceptive receptor upregulated in painful, inflamed tissues, and it can be imaged using the highly specific radioligand 18F-FTC-146 with PET.A 50-year-old woman with a 7-year history of refractory, left-knee pain of unknown origin was referred to our pain management team. Over the past several years, she had undergone multiple treatments, including a lateral retinacular release, radiofrequency ablation of a peripheral nerve, and physical therapy. While certain treatments provided partial relief, her pain would inevitably return to its original state. Using simultaneous positron emission tomography/magnetic resonance imaging (PET/MRI) with the novel radiotracer 18F-FTC-146, imaging showed increased focal uptake of 18F-FTC-146 in the intercondylar notch, corresponding to an irregular but equivocal lesion identified in the simultaneously acquired MRI. These imaging results prompted surgical removal of the lesion, which upon resection was identified as an inflamed, intraarticular synovial lipoma. Removal of the lesion relieved the patient's pain, and to date the pain has not recurred.We present a case of chronic, debilitating knee pain that resolved with surgery following identification of the pathology with a novel clinical molecular imaging approach that detects chronic pain generators at the molecular and cellular level. This approach has the potential to identify and localize pain-associated pathology in a variety of chronic pain syndromes.

  View details for PubMedID 30349360

• Loss of mu opioid receptor signaling in nociceptors, but not microglia, abrogates morphine tolerance without disrupting analgesia NATURE MEDICINE Corder, G., Tawfik, V. L., Wang, D., Sypek, E. I., Low, S. A., Dickinson, J. R., Sotoudeh, C., Clark, J. D., Barres, B. A., Bohlen, C. J., Scherrer, G. 2017; 23 (2): 164-173

  Abstract

  Opioid pain medications have detrimental side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). Tolerance and OIH counteract opioid analgesia and drive dose escalation. The cell types and receptors on which opioids act to initiate these maladaptive processes remain disputed, which has prevented the development of therapies to maximize and sustain opioid analgesic efficacy. We found that μ opioid receptors (MORs) expressed by primary afferent nociceptors initiate tolerance and OIH development. RNA sequencing and histological analysis revealed that MORs are expressed by nociceptors, but not by spinal microglia. Deletion of MORs specifically in nociceptors eliminated morphine tolerance, OIH and pronociceptive synaptic long-term potentiation without altering antinociception. Furthermore, we found that co-administration of methylnaltrexone bromide, a peripherally restricted MOR antagonist, was sufficient to abrogate morphine tolerance and OIH without diminishing antinociception in perioperative and chronic pain models. Collectively, our data support the idea that opioid agonists can be combined with peripheral MOR antagonists to limit analgesic tolerance and OIH.

  View details for DOI 10.1038/nm.4262

  View details for PubMedID 28092666

• Complex management of a patient with refractory primary erythromelalgia lacking a SCN9A mutation JOURNAL OF PAIN RESEARCH Low, S. A., Robbins, W., Tawfik, V. L. 2017; 10: 973-977

  Abstract

  A 41-year-old woman presented with burning and erythema in her extremities triggered by warmth and activity, which was relieved by applying ice. Extensive workup was consistent with adult-onset primary erythromelalgia (EM). Several pharmacological treatments were tried including local anesthetics, capsaicin, ziconotide, and dantrolene, all providing 24-48 hours of relief followed by symptom flare. Interventional therapies, including peripheral and sympathetic ganglion blocks, also failed. Thus far, clonidine and ketamine have been the only effective agents for our patient. Genetic testing was negative for an EM-associated mutation in the SCN9A gene, encoding the NaV1.7 sodium channel, suggesting a mutation in an alternate gene.

  View details for DOI 10.2147/JPR.S129661

  View details for Web of Science ID 000400133100003

  View details for PubMedCentralID PMC5414616

• (118) Physical and psychological predictors of dysfunction in complex regional pain syndrome (CRPS): a Collaborative Health Outcomes Information Registry (CHOIR) study. journal of pain Adelus, M., Sturgeon, J., RICO, T., Tawfik, V., Mackey, S. 2016; 17 (4S): S5-?

  View details for DOI 10.1016/j.jpain.2016.01.021

  View details for PubMedID 28162531

• (218) Characterization of patients with complex regional pain syndrome (CRPS) in a tertiary care pain management setting: A Collaborative Health Outcomes Information Registry (CHOIR) study. journal of pain Adelus, M., Sturgeon, J., RICO, T., Tawfik, V., Mackey, S. 2016; 17 (4S): S30-?

  View details for DOI 10.1016/j.jpain.2016.01.122

  View details for PubMedID 28162448

• Electrical Synapses High-speed Communication in the Maintenance of Neuropathic Pain ANESTHESIOLOGY Tawfik, V. L., Flood, P. 2016; 124 (1): 13-15

  View details for Web of Science ID 000366461300005

  View details for PubMedID 26566281

• Input- and Cell-Type-Specific Endocannabinoid-Dependent LTD in the Striatum. Cell reports Wu, Y., Kim, J., Tawfik, V. L., Lalchandani, R. R., Scherrer, G., Ding, J. B. 2015; 10 (1): 75-87

  Abstract

  Changes in basal ganglia plasticity at the corticostriatal and thalamostriatal levels are required for motor learning. Endocannabinoid-dependent long-term depression (eCB-LTD) is known to be a dominant form of synaptic plasticity expressed at these glutamatergic inputs; however, whether eCB-LTD can be induced at all inputs on all striatal neurons is still debatable. Using region-specific Cre mouse lines combined with optogenetic techniques, we directly investigated and distinguished between corticostriatal and thalamostriatal projections. We found that eCB-LTD was successfully induced at corticostriatal synapses, independent of postsynaptic striatal spiny projection neuron (SPN) subtype. Conversely, eCB-LTD was only nominally present at thalamostriatal synapses. This dichotomy was attributable to the minimal expression of cannabinoid type 1 (CB1) receptors on thalamostriatal terminals. Furthermore, coactivation of dopamine receptors on SPNs during LTD induction re-established SPN-subtype-dependent eCB-LTD. Altogether, our findings lay the groundwork for understanding corticostriatal and thalamostriatal synaptic plasticity and for striatal eCB-LTD in motor learning.

  View details for DOI 10.1016/j.celrep.2014.12.005

  View details for PubMedID 25543142

  View details for PubMedCentralID PMC4286501

• In vivo techniques to investigate the internalization profile of opioid receptors. Methods in molecular biology (Clifton, N.J.) Pradhan, A. A., Tawfik, V. L., Tipton, A. F., Scherrer, G. 2015; 1230: 87-104

  Abstract

  G-protein-coupled receptors (GPCRs) regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Receptor internalization is commonly observed following agonist binding and activation. Receptor trafficking events have been well characterized in cell systems, but the in vivo significance of GPCR internalization is still poorly understood. To address this issue, we have developed an innovative knock-in mouse model, where an opioid receptor is directly visible in vivo. These knockin mice express functional fluorescent delta opioid receptors (DOR-eGFP) in place of the endogenous receptor, and these receptors are expressed at physiological levels within their native environment. DOR-eGFP mice have proven to be an extraordinary tool in studying receptor neuroanatomy, real-time receptor trafficking in live neurons, and in vivo receptor internalization. We have used this animal model to determine the relationship between receptor trafficking in neurons and receptor function at a behavioral level. Here, we describe in detail the construction and characterization of this knockin mouse. We also outline how to use these mice to examine the behavioral consequences of agonist-specific trafficking at the delta opioid receptor. These techniques are potentially applicable to any GPCR, and highlight the powerful nature of this imaging tool.

  View details for DOI 10.1007/978-1-4939-1708-2_7

  View details for PubMedID 25293318

• Delta Opioid Receptors Presynaptically Regulate Cutaneous Mechanosensory Neuron Input to the Spinal Cord Dorsal Horn NEURON Bardoni, R., Tawfik, V. L., Wang, D., Francois, A., Solorzano, C., Shuster, S. A., Choudhury, P., Betelli, C., Cassidy, C., Smith, K., de Nooij, J. C., Mennicken, F., O'Donnell, D., Kieffer, B. L., Woodbury, C. J., Basbaum, A. I., MacDermott, A. B., Scherrer, G. 2014; 81 (6): 1312-1327

  Abstract

  Cutaneous mechanosensory neurons detect mechanical stimuli that generate touch and pain sensation. Although opioids are generally associated only with the control of pain, here we report that the opioid system in fact broadly regulates cutaneous mechanosensation, including touch. This function is predominantly subserved by the delta opioid receptor (DOR), which is expressed by myelinated mechanoreceptors that form Meissner corpuscles, Merkel cell-neurite complexes, and circumferential hair follicle endings. These afferents also include a small population of CGRP-expressing myelinated nociceptors that we now identify as the somatosensory neurons that coexpress mu and delta opioid receptors. We further demonstrate that DOR activation at the central terminals of myelinated mechanoreceptors depresses synaptic input to the spinal dorsal horn, via the inhibition of voltage-gated calcium channels. Collectively our results uncover a molecular mechanism by which opioids modulate cutaneous mechanosensation and provide a rationale for targeting DOR to alleviate injury-induced mechanical hypersensitivity.

  View details for DOI 10.1016/j.neuron.2014.01.044

  View details for Web of Science ID 000333326000012

  View details for PubMedID 24583022

• Perioperative interventions to reduce chronic postsurgical pain. Journal of reconstructive microsurgery Carroll, I., Hah, J., Mackey, S., Ottestad, E., Kong, J. T., Lahidji, S., Tawfik, V., Younger, J., Curtin, C. 2013; 29 (4): 213-222

  Abstract

  Approximately 10% of patients following a variety of surgeries develop chronic postsurgical pain. Reducing chronic postoperative pain is especially important to reconstructive surgeons because common operations such as breast and limb reconstruction have even higher risk for developing chronic postsurgical pain. Animal studies of posttraumatic nerve injury pain demonstrate that there is a critical time frame before and immediately after nerve injury in which specific interventions can reduce the incidence and intensity of chronic neuropathic pain behaviors-so called "preventative analgesia." In animal models, perineural local anesthetic, systemic intravenous local anesthetic, perineural clonidine, systemic gabapentin, systemic tricyclic antidepressants, and minocycline have each been shown to reduce pain behaviors days to weeks after treatment. The translation of this work to humans also suggests that brief perioperative interventions may protect patients from developing new chronic postsurgical pain. Recent clinical trial data show that there is an opportunity during the perioperative period to dramatically reduce the incidence and severity of chronic postsurgical pain. The surgeon, working with the anesthesiologist, has the ability to modify both early and chronic postoperative pain by implementing an evidence-based preventative analgesia plan.

  View details for DOI 10.1055/s-0032-1329921

  View details for PubMedID 23463498

• Perioperative Interventions to Reduce Chronic Postsurgical Pain JOURNAL OF RECONSTRUCTIVE MICROSURGERY Carroll, I., Hah, J., Mackey, S., Ottestad, E., Kong, J. T., Lahidji, S., Tawfik, V., Younger, J., Curtin, C. 2013; 29 (4): 213-222

  Abstract

  Approximately 10% of patients following a variety of surgeries develop chronic postsurgical pain. Reducing chronic postoperative pain is especially important to reconstructive surgeons because common operations such as breast and limb reconstruction have even higher risk for developing chronic postsurgical pain. Animal studies of posttraumatic nerve injury pain demonstrate that there is a critical time frame before and immediately after nerve injury in which specific interventions can reduce the incidence and intensity of chronic neuropathic pain behaviors-so called "preventative analgesia." In animal models, perineural local anesthetic, systemic intravenous local anesthetic, perineural clonidine, systemic gabapentin, systemic tricyclic antidepressants, and minocycline have each been shown to reduce pain behaviors days to weeks after treatment. The translation of this work to humans also suggests that brief perioperative interventions may protect patients from developing new chronic postsurgical pain. Recent clinical trial data show that there is an opportunity during the perioperative period to dramatically reduce the incidence and severity of chronic postsurgical pain. The surgeon, working with the anesthesiologist, has the ability to modify both early and chronic postoperative pain by implementing an evidence-based preventative analgesia plan.

  View details for DOI 10.1055/s-0032-1329921

  View details for Web of Science ID 000317597000001

  View details for PubMedID 23463498

• Deep Brain Stimulation Results in Local Glutamate and Adenosine Release: Investigation Into the Role of Astrocytes NEUROSURGERY Tawfik, V. L., Chang, S., Hitti, F. L., Roberts, D. W., Leiter, J. C., Jovanovic, S., Lee, K. H. 2010; 67 (2): 367-375

  Abstract

  Several neurological disorders are treated with deep brain stimulation; however, the mechanism underlying its ability to abolish oscillatory phenomena associated with diseases as diverse as Parkinson's disease and epilepsy remain largely unknown.To investigate the role of specific neurotransmitters in deep brain stimulation and determine the role of non-neuronal cells in its mechanism of action.We used the ferret thalamic slice preparation in vitro, which exhibits spontaneous spindle oscillations, to determine the effect of high-frequency stimulation on neurotransmitter release. We then performed experiments using an in vitro astrocyte culture to investigate the role of glial transmitter release in high-frequency stimulation-mediated abolishment of spindle oscillations.In this series of experiments, we demonstrated that glutamate and adenosine release in ferret slices was able to abolish spontaneous spindle oscillations. The glutamate release was still evoked in the presence of the Na channel blocker tetrodotoxin, but was eliminated with the vesicular H-ATPase inhibitor bafilomycin and the calcium chelator 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester. Furthermore, electrical stimulation of purified primary astrocytic cultures was able to evoke intracellular calcium transients and glutamate release, and bath application of 2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester inhibited glutamate release in this setting.Vesicular astrocytic neurotransmitter release may be an important mechanism by which deep brain stimulation is able to achieve clinical benefits.

  View details for DOI 10.1227/01.NEU.0000371988.73620.4C

  View details for Web of Science ID 000280105800032

  View details for PubMedID 20644423

• Propentofylline-induced astrocyte modulation leads to alterations in glial glutamate promoter activation following spinal nerve transection NEUROSCIENCE Tawfik, V. L., Regan, M. R., Haenggeli, C., Lacroix-Fralish, M. L., Nutile-McMenemy, N., Perez, N., Rothstein, J. D., DeLeo, J. A. 2008; 152 (4): 1086-1092

  Abstract

  We have previously shown that the atypical methylxanthine, propentofylline, reduces mechanical allodynia after peripheral nerve transection in a rodent model of neuropathy. In the present study, we sought to determine whether propentofylline-induced glial modulation alters spinal glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in vivo, which may contribute to reduced behavioral hypersensitivity after nerve injury. In order to specifically examine the expression of the spinal glutamate transporters, a novel line of double transgenic GLT-1-enhanced green fluorescent protein (eGFP)/GLAST-Discosoma Red (DsRed) promoter mice was used. Adult mice received propentofylline (10 mg/kg) or saline via i.p. injection starting 1 h prior to L5-spinal nerve transection and then daily for 12 days. Mice receiving saline exhibited punctate expression of both eGFP (GLT-1 promoter activation) and DsRed (GLAST promoter activation) in the dorsal horn of the spinal cord, which was decreased ipsilateral to nerve injury on day 12. Propentofylline administration reinstated promoter activation on the injured side as evidenced by an equal number of eGFP (GLT-1) and DsRed (GLAST) puncta in both dorsal horns. As demonstrated in previous studies, propentofylline induced a concomitant reversal of L5 spinal nerve transection-induced expression of glial fibrillary acidic protein (GFAP). The ability of propentofylline to alter glial glutamate transporters highlights the importance of controlling aberrant glial activation in neuropathic pain and suggests one possible mechanism for the anti-allodynic action of this drug.

  View details for DOI 10.1016/j.neuroscience.2008.01.065

  View details for Web of Science ID 000255152800022

  View details for PubMedID 18358622

• Neuregulin 1 is a pronociceptive cytokine that is regulated by progesterone in the spinal cord: Implications for sex specific pain modulation EUROPEAN JOURNAL OF PAIN LaCroix-Fralish, M. L., Tawfik, V. L., Nutile-McMenemy, N., DeLeo, J. A. 2008; 12 (1): 94-103

  Abstract

  Sex differences in the magnitude of response to thermal and tactile stimuli have been demonstrated in both clinical and animal studies. Females typically display lower threshold responses to painful stimuli as compared to males. We have previously observed sexually dimorphic expression of the growth factor, neuregulin 1 (NRG1) following L5 nerve root ligation (LR) in male and female rats. In the present study, we sought to determine which gonadal hormones were involved in regulating NRG1 expression following L5 nerve root ligation. We observed that expression of NRG1 mRNA and the neuregulin receptors, ErbB2 and ErbB4 in the lumbar spinal cord was facilitated by the presence of progesterone in female rats following L5 nerve root ligation. An increase in NRG1 protein and NRG1 immunoreactivity was also observed in the ipsilateral spinal cord of progesterone treated female rats as compared to ovariectomized female rats and male rats at day 14 following LR. NRG1 immunoreactivity was equally colocalized with either the astrocytic marker, GFAP, and with NeuN labeled neurons 14days following L5 nerve root ligation. Intrathecal administration of recombinant NRG1-beta1 protein significantly decreased the hindpaw tactile withdrawal threshold in male rats, ovariectomized female rats, and progesterone treated female rats. These results demonstrate a role for progesterone-dependent regulation of glial and/or neuronal neuregulin 1 in female rats in mediating sex differences in nociception. Furthermore, our results suggest that NRG1 may be involved in central sensitization during the maintenance phase, but not in the initiation of persistent pain in female rats.

  View details for DOI 10.1016/j.ejpain.2007.03.010

  View details for Web of Science ID 000251934200012

  View details for PubMedID 17459743

• Intracranial self-stimulation of the dorsal raphe sensitizes psychostimulant locomotion BEHAVIORAL NEUROSCIENCE Boye, S. M., Grant, R. J., Tawfik, V. L. 2007; 121 (3): 550-558

  Abstract

  The authors hypothesized that repeated rewarding electrical stimulation of the dorsal raphe can produce behavioral sensitization to psychostimulants. Groups of male rats were implanted with a stimulation electrode and preexposed to brain stimulation at parameters set to equate rewarding effectiveness across rats. Control groups were implanted with an electrode but never stimulated, or not implanted at all. Twenty-four hours after the 12th self-stimulation session, all groups were challenged with amphetamine (0.5 mg/kg, ip), nicotine (0.2 mg/kg, sc), or saline, and locomotor activity was measured for 1 hr. Locomotor responses to amphetamine and to nicotine were significantly greater in rats preexposed to brain stimulation. These findings suggest at least partial overlap of underlying substrates. ((c) 2007 APA, all rights reserved).

  View details for DOI 10.1037/0735-7044.121.3.550

  View details for Web of Science ID 000247359300011

  View details for PubMedID 17592946

• Efficacy of propentofylline, a glial modulating agent, on existing mechanical allodynia following peripheral nerve injury BRAIN BEHAVIOR AND IMMUNITY Tawfik, V. L., Nutile-McMenemy, N., LaCroix-Fralish, M. L., DeLeo, J. A. 2007; 21 (2): 238-246

  Abstract

  Increasing evidence points to a role for spinal neuroimmune dysregulation (glial cell activation and cytokine expression) in the pathogenesis of chronic pain. Suppression of astrocytic and microglial activation with the methylxanthine derivative, propentofylline, pre-emptively attenuates the development of nerve injury-induced allodynia. Currently, we investigated the ability of systemic propentofylline to reverse existing, long-term allodynia after nerve injury--a clinically relevant paradigm. Rats received L5 spinal nerve transection or sham surgery and the development of mechanical allodynia was assessed daily for 2 weeks, at which time injured rats exhibited robust responses to non-noxious von Frey filaments. On days 14-27, rats received either saline or 101 mg/kg propentofylline by intraperitoneal (i.p.) injection. On day 28 or 42 (after a 14-day drug washout period), lumbar spinal cord sections were processed for assessment of astrocytic glial fibrillary acidic protein (GFAP) and microglial OX-42 (antibody against CR3/CD11b). Propentofylline treatment to nerve injured rats resulted in significant reversal of allodynia that lasted throughout the 14-day washout period. Spinal microglial activation was observed at days 28 and 42 post-injury at the protein level, in the absence of mRNA level changes. Less robust increases in GFAP immunoreactivity were observed at days 28 and 42 post-transection. Interestingly, propentofylline treatment suppressed microglial activation at both time points in this paradigm. Taken together, our results highlight the clinical potential of the glial modulating agent, propentofylline, for the treatment of neuropathic pain as well as a role for microglia in the long-term maintenance of allodynia.

  View details for DOI 10.1016/j.bbi.2006.07.001

  View details for Web of Science ID 000243896100014

  View details for PubMedID 16949251

• Induction of astrocyte differentiation by propentofylline increases glutamate transporter expression in vitro: Heterogeneity of the quiescent phenotype GLIA Tawfik, V. L., LaCroix-Fralish, M. L., Bercury, K. K., Nutile-McMenemy, N., Harris, B. T., DeLeo, J. A. 2006; 54 (3): 193-203

  Abstract

  Reactive astrocytes display decreased glutamate transporters, such as GLT-1, and as a result synaptic glutamate clearance is impaired. In addition, these activated astrocytes are immunocompetent and release algesic mediators that can sensitize neurons in the spinal cord. Currently, we evaluated the effect of propentofylline (PPF), an experimental antiallodynic agent, on the phenotype and glutamate transporter expression of astrocytes. Primary astrocyte cultures, which represent an activated phenotype with a polygonal morphology and low GLT-1 expression, were treated for 3 or 7 days with 10, 100, or 1,000 microM PPF or dibutyryl-cAMP (db-cAMP), a known inducer of GLT-1 expression. PPF dose-dependently induced astrocytes to display a mature phenotype, with elongated processes and a stellate shape, as well as increased GLT-1 and GLAST immunoreactivity, similar to that seen with db-cAMP. Real time RT-PCR and Western blot analysis clearly demonstrated that PPF caused a potent dose-dependent induction of GLT-1 and GLAST mRNA and protein in these astrocytes. Importantly, the observed increase in glutamate transporters was found to have a functional effect, with significantly enhanced glutamate uptake in astrocytes treated with 100 or 1,000 microM PPF that was sensitive to dihydrokainate inhibition, suggesting it is GLT-1 mediated. Finally, the effect of PPF on lipopolysaccharide-induced chemokine release was investigated. Interestingly, PPF was able to dampen both MCP-1 (CCL2) and MIP-2 (CXCL2) release from astrocytes while db-cAMP significantly enhanced this chemokine expression. These findings suggest that PPF is capable of differentiating astrocytes to a homeostatic, mature phenotype, competent for glutamate clearance and distinct from that induced by db-cAMP.

  View details for DOI 10.1002/glia.20365

  View details for Web of Science ID 000239351600005

  View details for PubMedID 16819765

• Differential spinal cord gene expression in rodent models of radicular and neuropathic pain ANESTHESIOLOGY LaCroix-Fralish, M. L., Tawfik, V. L., Tanga, F. Y., Spratt, K. F., DeLeo, J. A. 2006; 104 (6): 1283-1292

  Abstract

  Neuropathic pain and radicular low back pain both have a major impact on human health worldwide. Microarray gene analysis on central nervous system tissues holds great promise for discovering novel targets for persistent pain modulation.Rat models of lumbar radiculopathy (L5 nerve root ligation) and neuropathy (L5 spinal nerve transection) were used for these studies. The authors measured mechanical allodynia followed by analysis of global gene expression in the lumbar spinal cord at two time points (7 days and 14 days) after surgery using the Affymetrix RAE230A GeneChip(R) (Santa Clara, CA). The expression patterns of several genes of interest were subsequently confirmed using real-time reverse transcriptase polymerase chain reaction.The authors observed similarly robust mechanical allodynia in both models. Second, they observed significant differences in lumbar spinal cord gene expression across chronic pain models. There was little overlap between genes altered in each injury model, suggesting that the site and type of injury produce distinct spinal mechanisms mediating the observed mechanical allodynia. The authors further confirmed a subset of the genes using reverse transcriptase polymerase chain reaction and identified several genes as either neuropathy-associated genes or radiculopathy-associated genes.These two models of persistent pain produce similar allodynic outcomes but produce differential gene expression. These results suggest that diverging mechanisms lead to a common behavioral outcome in these pain models. Furthermore, these distinct pathophysiologic mechanisms in neuropathic versus radicular pain may implicate unique drug therapies for these types of chronic pain syndromes.

  View details for Web of Science ID 000237869500024

  View details for PubMedID 16732101

• The tetrapartite synapse: Path to CNS sensitization and chronic pain PAIN de Leo, J. A., Tawfik, V. L., LaCroix-Fralish, M. L. 2006; 122 (1-2): 17-21

  View details for DOI 10.1016/j.pain.2006.02.034

  View details for Web of Science ID 000237549100007

  View details for PubMedID 16564626

• Progesterone mediates gonadal hormone differences in tactile and thermal hypersensitivity following L5 nerve root ligation in female rats NEUROSCIENCE Lacroix-Fralish, M. L., Tawfik, V. L., Nutile-McMenemy, N., DeLeo, J. A. 2006; 138 (2): 601-608

  Abstract

  Sex differences in the magnitude of response to thermal and tactile stimuli have been demonstrated in both clinical and animal studies. Female rats typically display lower thresholds to painful stimuli and display more robust responses following nerve injury as compared with males. There is a body of evidence implicating the sex hormones in mediating this sex difference. In the present study, we sought to determine which gonadal hormones were involved in mediating the observed female hypersensitivity in female rats both prior to and following experimental nerve root injury using a chronic hormone replacement paradigm. Female rats were ovariectomized and hormone pellets containing 17beta-estradiol, progesterone (P), 17beta-estradiol+progesterone or placebo were implanted s.c. Our results demonstrate that only the group of female rats that received progesterone alone maintained the hypersensitive phenotype following ovariectomy, compared with gonadally intact male rats. This result was observed both in response to thermal stimuli in non-injured female rats and to thermal and tactile stimuli following L5 nerve root ligation, a model of low back pain associated with lumbar radiculopathy. Postmortem analysis of serum gonadal hormone concentrations demonstrates that the hormonal manipulations were successful and the exogenous hormones were similar to physiological levels observed in the sham-ovariectomized controls. Taken together, these results demonstrate the critical role for progesterone in mediating enhanced female tactile and thermal hypersensitivity following L5 nerve root ligation.

  View details for DOI 10.1016/j.neuroscience.2005.11.048

  View details for Web of Science ID 000236046100021

  View details for PubMedID 16413124

• Sex differences in lumbar spinal cord gene expression following experimental lumbar radiculopathy JOURNAL OF MOLECULAR NEUROSCIENCE LaCroix-Fralish, M. L., Tawfik, V. L., Spratt, K. F., DeLeo, J. A. 2006; 30 (3): 283-295

  Abstract

  Considerable evidence indicates that there are sex-related differences in clinical and experimental pain sensitivity. In the present study, we sought to determine what genes were expressed in the spinal cord in a sexually dimorphic manner. We first analyzed global gene expression in the lumbar spinal cord of uninjured male and female rats using the Affymetrix RAE230A GeneChip platform in order to identify genes that are selectively expressed in male and female rats at a basal level. We subsequently analyzed global gene expression in the lumbar spinal cord of male and female rats at two time points (7 days and 14 d) following a rodent model of lumbar radiculopathy (L5 nerve root ligation) in order to determine what genes were regulated in a sexually dimorphic manner following nerve root injury. We utilized a linear regression analysis method to identify genes that were significantly different from the corresponding sham surgical controls. The expression patterns of several genes of interest were subsequently confirmed using RT-PCR. Our findings demonstrate significant differences in lumbar spinal cord gene expression in both uninjured and injured (L5 nerve root ligation) male and female rats. Further confirmation of a subset of the genes identified Neuregulin 1 and its high affinity receptor, ErbB4, Tachykinin 1, and Metabotropic glutamate receptor 6 as female specific genes upregulated following L5 nerve root injury. These findings provide several target genes for further study that may elucidate the neurochemical mechanisms underlying sex differences in pain sensitivity and lead to improved treatments for chronic pain syndromes.

  View details for DOI 10.1385/JMN/30:03:283

  View details for Web of Science ID 000243782200004

  View details for PubMedID 17401154

• Differential regulation of neuregulin 1 expression by progesterone in astrocytes and neurons NEURON GLIA BIOLOGY LaCroix-Fralish, M. L., Tawfik, V. L., Nutile-McMenemy, N., Harris, B. T., DeLeo, J. A. 2006; 2: 227-234

  Abstract

  Glial-neuronal interactions are crucial processes in neuromodulation and synaptic plasticity. The neuregulin 1 family of growth and differentiation factors have been implicated as bidirectional signaling molecules that are involved in mediating some of these interactions. We have shown previously that neuregulin 1 expression is regulated by the gonadal hormones progesterone and 17beta-estradiol in the CNS, which might represent a novel, indirect mechanism of the neuromodulatory actions of these gonadal hormones. In the present study, we sought to determine the effects of progesterone and 17beta-estradiol on neuregulin 1 expression in rat cortical astrocytes and neurons in vitro. We observed that progesterone increased the expression of neuregulin 1 mRNA and protein in a dose-dependent manner in cultured astrocytes, which was blocked by the progesterone receptor antagonist RU-486. In contrast, 17beta-estradiol did not increase either neuregulin 1 mRNA or protein in astrocytes. We observed no effect of either progesterone or 17beta-estradiol on neuregulin 1 mRNA and protein in rat cortical neurons in vitro. Finally, we observed that treatment of cortical neurons with recombinant NRG1-beta1 caused PSD-95 to localize in puncta similar to that observed following treatment with astrocyte-conditioned medium. These results demonstrate that progesterone regulates neuregulin 1 expression, principally in astrocytes. This might represent a novel mechanism of progesterone-mediated modulation of neurotransmission through the regulation of astrocyte-derived neuregulin 1.

  View details for DOI 10.1017/S1740925X07000385

  View details for Web of Science ID 000248510100002

  View details for PubMedID 18049715

• Safety issues concerning the medical use of cannabis and cannabinoids. Pain research & management Ware, M. A., Tawfik, V. L. 2005; 10: 31A-7A

  Abstract

  Safety issues are a major barrier to the use of cannabis and cannabinoid medications for clinical purposes. Information on the safety of herbal cannabis may be derived from studies of recreational cannabis use, but cannabis exposure and effects may differ widely between medical and recreational cannabis users. Standardized, quality-controlled cannabinoid products are available in Canada, and safety profiles of approved medications are available through the Canadian formulary. In the present article, the evidence behind major safety issues related to cannabis use is summarized, with the aim of promoting informed dialogue between physicians and patients in whom cannabinoid therapy is being considered. Caution is advised in interpreting these data, because clinical experience with cannabinoid use is in the early stages. There is a need for long-term safety monitoring of patients using cannabinoids for a wide variety of conditions, to further guide therapeutic decisions and public policy.

  View details for PubMedID 16237480

• Transcriptional and translational regulation of glial activation by morphine in a rodent model of neuropathic pain JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Tawfik, V. L., Lacroix-Fralish, M. L., Nutile-McMeneny, N., DeLeo, J. A. 2005; 313 (3): 1239-1247

  Abstract

  Glial cells function in maintenance of homeostasis as well as in pathophysiology. In this study, we determined the time course of spinal glial cell activation during the development of morphine analgesic tolerance in an L5 spinal nerve transection rodent model of neuropathic pain. We also sought to assess whether the method of morphine administration affected neuroimmune activation at the levels of transcription and translation. Rats received L5 spinal nerve transection or no surgery on day 0. On day 6 post-transection, osmotic minipumps were implanted to deliver saline or morphine s.c. (1 or 10 mg/kg) or i.t. (5 or 20 nmol/h). Mechanical allodynia developed immediately after spinal nerve transection; this hypersensitivity was reversed with both low- and high-dose morphine by either route. Tolerance to antiallodynia developed after 3 days of i.t. morphine and after 6 days of s.c. morphine, indicating hastened tolerance following i.t. delivery. Analysis of mRNA revealed that s.c. morphine treatment did not lead to increases in glial activation markers. In contrast, i.t. morphine caused a biphasic alteration in glial fibrillary acidic protein (GFAP) and integrin alpha M mRNA. Protein levels for GFAP were elevated after s.c. and i.t. administration of morphine; however, induction was further enhanced in the latter group. Here, we show for the first time that there is differential recruitment of transcriptional and translational mechanisms of glial activation by systemic and i.t. morphine. Furthermore, we suggest that enhanced neuroimmune activation after i.t. dosing contributes to the hastened development of analgesic tolerance seen in these animals.

  View details for DOI 10.1124/jpet.104.082420

  View details for Web of Science ID 000229203200033

  View details for PubMedID 15743926

• The organizational and activational effects of sex hormones on tactile and thermal hypersensitivity following lumbar nerve root injury in male and female rats PAIN Lacroix-Fralish, M. L., Tawfik, V. L., DeLeo, J. A. 2005; 114 (1-2): 71-80

  Abstract

  Considerable evidence exists for sex differences in human pain sensitivity. Women typically report a higher incidence of various painful conditions and report that the conditions are more painful when compared to men. In the present study, we sought to determine whether sex differences in pain sensitivity are observed using a lumbar radiculopathy model of low back pain in the rat and whether removal or alteration of gonadal hormones at specific timepoints can modulate these sex differences. Pubertal and adult male and female Sprague-Dawley rats were castrated 2 or 6 weeks prior to L5 nerve root injury to determine the activational hormonal effects. In a separate study, neonatal male and female Sprague-Dawley rats were either castrated or injected with testosterone, respectively, on postnatal day one to determine the organizational effects of gonadal hormones on L5 nerve root injury-induced behavioral hypersensitivity. Our results demonstrate that there was a statistically significant sex difference in the magnitude of mechanical allodynia and thermal hyperalgesia following experimentally induced radiculopathy in the rat: females demonstrated decreased thresholds to tactile and thermal stimuli as compared to males. Furthermore, the enhanced female hypersensitivity was reversed in pubertal and adult animals ovariectomized 6 weeks, but not 2 weeks prior to L5 nerve root injury. Our results demonstrate that the activational effects of gonadal hormones mediate the enhanced female tactile and thermal hypersensitivity following L5 nerve root injury. These results suggest that manipulation of gonadal hormones may be a potential source for novel therapies for chronic pain in women.

  View details for DOI 10.1016/j.pain.2004.12.006

  View details for Web of Science ID 000227683500010

  View details for PubMedID 15733633

• Neuroimmune activation and neuroinflammation in chronic pain and opioid tolerance/hyperalgesia. Neuroscientist DeLeo, J. A., Tanga, F. Y., Tawfik, V. L. 2004; 10 (1): 40-52