Treatment with shCCL20-CCR6 nanodendriplexes and human mesenchymal stem cell therapy improves pathology in mice with repeated traumatic brain injury.

Traumatic brain injury (TBI) is a devastating neurological disorder, although the underlying pathophysiology is poorly understood. TBI causes blood-brain barrier (BBB) disruption, immune cell trafficking, neuroinflammation and neurodegeneration. CCL20 is an important chemokine mediating neuroinflammation. Human mesenchymal stem cell (hMSC) therapy is a promising regenerative approach but the inflammatory microenvironment in the brain tends to decrease the efficacy of the hMSC transplantation. Reducing the inflammation prior to hMSC therapy improves the outcome. We developed a combined nano-cell therapy by using dendrimers complexed with plasmids (dendriplexes) targeting CCL20 and its sole receptor CCR6 to reduce inflammation followed by hMSC transplantation. Treatment of TBI mice with shRNA conjugated dendriplexes followed by hMSC administration downregulated the inflammatory markers and significantly increased brain-derived neurotrophic factor (BDNF) expression in the cerebral cortex indicating future possible neurogenesis and improved behavioral deficits. Taken together, this nano-cell therapy ameliorates neuroinflammation and promotes brain tissue repair after TBI.

Prevalence and risk factors of obstetric fistula: implementation of a need-based preventive action plan in a South-eastern rural community of India.

BACKGROUND: The study was conducted to estimate the prevalence and risk factors of obstetric fistula in the rural area of the south eastern community of India and the training of community health workers for its prevention. METHODS: A population-based cross-sectional analytical study was conducted in the south eastern rural community of India. A total of 3939 women were included in the study and Probability proportional to size sampling was used in the study. Frequency distribution and logistic regression were computed to analyse the data using STATA version 11.2. RESULTS: Out of 3939 participants interviewed, 23.7% women reported obstetric fistula symptoms whereas after clinical diagnosis and speculum examination the obstetric morbidity pattern was: Obstetric fistula 0.3%, stress urinary incontinence 20.0%, pelvic inflammatory diseases 1.2%, uterine prolapse 1.4% and urinary tract infection 3.8%. The awareness level of the rural women regarding the obstetric fistula was assessed by a structured knowledge questionnaire and found to be very poor, hence community based fistula training was implemented among community health workers as a health system based strategy for its prevention. Obstetric fistula found to be more prevalent among women of poor educational level, low socioeconomic status, less no of antenatal visits, delay in accessing the emergency obstetric care and prolonged labour (p ≤ 0.05). CONCLUSION: Finding of the study indicated that the prevalence and risk of developing obstetric fistula was associated with less number of antenatal visits, prolonged labour, delay in timely intervention, delay in accessing emergency obstetric care and more number of movements from home to the delivery place. Finally, our study suggests that emphasis needs to be placed on training of community health workers to facilitate early screening for identification and referral of women with obstetric fistula.

Potential of mesenchymal stem cells alone, or in combination, to treat traumatic brain injury.

Traumatic brain injury (TBI) causes death and disability in the United States and around the world. The traumatic insult causes the mechanical injury of the brain and primary cellular death. While a comprehensive pathological mechanism of TBI is still lacking, the focus of the TBI research is concentrated on understanding the pathophysiology and developing suitable therapeutic approaches. Given the complexities in pathophysiology involving interconnected immunologic, inflammatory, and neurological cascades occurring after TBI, the therapies directed to a single mechanism fail in the clinical trials. This has led to the development of the paradigm of a combination therapeutic approach against TBI. While there are no drugs available for the treatment of TBI, stem cell therapy has shown promising results in preclinical studies. But, the success of the therapy depends on the survival of the stem cells, which are limited by several factors including route of administration, health of the administered cells, and inflammatory microenvironment of the injured brain. Reducing the inflammation prior to cell administration may provide a better outcome of cell therapy following TBI. This review is focused on different therapeutic approaches of TBI and the present status of the clinical trials.

Impact of Community-Based Continuous Training on Promoting Birth Preparedness and Pregnancy Outcome in Rural Odisha, India: An Interventional Study.

BACKGROUND: Birth preparedness and complication readiness extends the maternal and neonatal health continuum of care and thus contributes to one of the important tools for pregnant women to experience better pregnancy outcome, strengthening family and community health, creating space for other interventions. The present study aimed to evaluate community-based birth preparedness and complication readiness training on pregnancy outcome. METHOD: The study adopted a quasi-experimental time series only one experimental design which was conducted in rural south-eastern India for 1 year among the reproductive age group 15-49 years (≤ 24 weeks pregnancy), and cases were followed up till postnatal period. A standardized birth preparedness assessment index (BPAI) was used to assess preparedness level of respondents. Community-based continuous training (CBCT) was introduced, and its effect was measured on birth preparedness level, involvement of family and their pregnancy outcomes. RESULT: CBCT interventional program was effective in promoting positive behaviors on birth preparedness and complication readiness as per BPAI: 13% of women were at level 1, 15% at level 2, 19% at level 3, 49% participants were at 4th level and 5% were at 5th level which represented the best level of preparedness for their present delivery. Pregnant mothers who completed their antenatal visits and were well prepared for delivery were found to be having two times favorable pregnancy outcome than those who had not (OR 2.79). CONCLUSION: BPCR intervention strategy can be utilized as a timely and effective community action plan for ensuring a favorable pregnancy outcome.

Pioglitazone treatment prior to transplantation improves the efficacy of human mesenchymal stem cells after traumatic brain injury in rats.

Traumatic brain injury is a leading cause of death and disability around the world. So far, drugs are not available to repair brain damage. Human mesenchymal stem cell (hMSC) transplantation therapy is a promising approach, although the inflammatory microenvironment of the injured brain affects the efficacy of transplanted hMSCs. We hypothesize that reducing the inflammation in the cerebral microenvironment by reducing pro-inflammatory chemokines prior to hMSC administration will improve the efficacy of hMSC therapy. In a rat model of lateral fluid percussion injury, combined pioglitazone (PG) and hMSC (combination) treatment showed less anxiety-like behavior and improved sensorimotor responses to a noxious cold stimulus. Significant reduction in brain lesion volume, neurodegeneration, microgliosis and astrogliosis were observed after combination treatment. TBI induced expression of inflammatory chemokine CCL20 and IL1-ß were significantly decreased in the combination treatment group. Combination treatment significantly increased brain-derived neurotrophic factor (BDNF) level and subventricular zone (SVZ) neurogenesis. Taken together, reducing proinflammatory cytokine expression in the cerebral tissues after TBI by PG administration and prior to hMSC therapy improves the outcome of the therapy in which BDNF could have a role.

Mesenchymal stem cell therapy for the treatment of traumatic brain injury: progress and prospects.

Traumatic brain injury (TBI) is a major cause of injury-related mortality and morbidity in the USA and around the world. The survivors may suffer from cognitive and memory deficits, vision and hearing loss, movement disorders, and different psychological problems. The primary insult causes neuronal damage and activates astrocytes and microglia which evokes immune responses causing further damage to the brain. Clinical trials of drugs to recover the neuronal loss are not very successful. Regenerative approaches for TBI using mesenchymal stem cells (MSCs) seem promising. Results of preclinical research have shown that transplantation of MSCs reduced secondary neurodegeneration and neuroinflammation, promoted neurogenesis and angiogenesis, and improved functional outcome in the experimental animals. The functional improvement is not necessarily related to cell engraftment; rather, immunomodulation by molecular factors secreted by MSCs is responsible for the beneficial effects of this therapy. However, MSC therapy has a few drawbacks including tumor formation, which can be avoided by the use of MSC-derived exosomes. This review has focused on the research works published in the field of regenerative therapy using MSCs after TBI and its future direction.

CCL20-CCR6 axis modulated traumatic brain injury-induced visual pathologies.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of death and disability in the USA and the world; it constitutes 30% of injury-related deaths (Taylor et al., MMWR Surveill Summ 66:1-16, 2017). Contact sports athletes often experience repetitive TBI (rTBI), which exerts a cumulative effect later in life. Visual impairment is a common after-effect of TBI. Previously, we have shown that C-C chemokine 20 (CCL20) plays a critical role in neurodegeneration and inflammation following TBI (Das et al., J Neuroinflammation 8:148, 2011). C-C chemokine receptor 6 (CCR6) is the only receptor that CCL20 interacts with. The objective of the present study was to investigate the role of CCL20-CCR6 axis in mediating rTBI-induced visual dysfunction (TVD). METHODS: Wild type (WT) or CCR6 knock out (CCR6-/-) mice were subjected to closed head rTBI. Pioglitazone (PG) is a peroxisome proliferator-activated receptor γ (PPARγ) agonist which downregulates CCL20 production. Subsets of WT mice were treated with PG following final rTBI. A subset of mice was also treated with anti-CCL20 antibody to neutralize the CCL20 produced after rTBI. Histopathological assessments were performed to show cerebral pathologies, retinal pathologies, and inflammatory changes induced by rTBI. RESULTS: rTBI induced cerebral neurodegeneration, retinal degeneration, microgliosis, astrogliosis, and CCL20 expression. CCR6-/- mice showed reduced retinal degeneration, microgliosis, and inflammation. Treatment with CCL20 neutralization antibody or PG showed reduced CCL20 expression along with reduced retinal degeneration and inflammation. rTBI-induced GFAP-positive glial activation in the optic nerve was not affected by knocking out CCR6. CONCLUSION: The present data indicate that rTBI-induced retinal pathology is mediated at least in part by CCL20 in a CCR6-dependent manner.

Obstetric fistula: A challenge to public health.

Obstetric fistula (OF) is one of the most important consequences of a prolonged obstructed labor, a big issue for low-income countries (LICs) like India. The objective is to identify and explore the knowledge regarding OF as a public health problem in LICs from peer review literature. The PubMed, Google Scholar, and Science Direct databases were searched to identify the prevalence, risk factors, and management of OF in LICs. Quantitative evidence-based paper reviewed. Twenty-seven articles met the inclusion criteria. The 15 provided population-based OF prevalence data of OF and 12 provided risk factors and social causes of OF rates associated with the birth that caused an OF. OF has one of the big public health problems. There is a lack of scientific research on the prevalence and risk factors of OF in LICs. This review helps to eradicate or alleviate the problem of OF in LICs like India.

Vision impairment after traumatic brain injury: present knowledge and future directions.

Traumatic brain injury (TBI) is a major cause of mortality and morbidity in the USA as well as in the world. As a result of TBI, the visual system is also affected often causing complete or partial visual loss, which in turn affects the quality of life. It may also lead to ocular motor dysfunction, defective accommodation, and impaired visual perception. As a part of the therapeutic strategy, early rehabilitative optometric intervention is important. Orthoptic therapy, medication, stem cell therapy, motor and attention trainings are the available treatment options. Gene therapy is one of the most promising emerging strategies. Use of state-of-the-art nanomedicine approaches to deliver drug(s) and/or gene(s) might enhance the therapeutic efficacy of the present and future modalities. More research is needed in these fields to improve the outcome of this debilitating condition. This review focuses on different visual pathologies caused by TBI, advances in pre-clinical and clinical research, and available treatment options.

Sertoli Cells Loaded with Doxorubicin in Lipid Micelles Reduced Tumor Burden and Dox-Induced Toxicity.

The toxic side effects of doxorubicin (Dox) limit its long-term use as a lung cancer chemotherapeutic. Additionally, drug delivery to the deep lung is challenging. To address these challenges, isolated rat Sertoli cells (SCs) were preloaded with Dox conjugated to lipid micelle nanoparticles (SC-DLMNs) and delivered to mouse lungs. These immunocompetent cells, when injected intravenously, travel to the lung, deliver the payload, and get cleared by the system quickly without causing any adverse reaction. We observed that SC-DLMNs effectively treated Lewis lung carcinoma 1-induced lung tumors in mice and the drug efficacy was comparable to SC-Dox treatment. Mice treated with SC-DLMNs also showed significantly less toxicity compared to those treated with SC-Dox. The encapsulation of Dox in lipid micelle nanoparticles reduced the toxicity of Dox and the SC-based delivery method ensured drug delivery to the deep lung without evoking any immune response. Taken together, these results provide a novel SC-based nanoparticle drug delivery method for improved therapeutic outcome of cardiotoxic antilung cancer drugs.

QUANTITATIVE MORPHOLOGICAL AND MOLECULAR PATHOLOGY OF THE HUMAN THYMUS CORRELATE WITH INFANT CAUSE OF DEATH.

The objective of this study was to investigate and quantify the morphological and molecular changes in the thymus for common causes of human infant death. Thymic architecture and molecular changes apparent in human infant head trauma victims were assessed by microscopy and quantified by image analysis of digital whole slide images. Thymuses from victims of SIDS and suffocated infants displaying normal thymus architecture were used for comparison. Molecular expression of proliferation and serotonin receptor and transporter protein markers was evaluated. Duplicate morphological and molecular studies of rodent thymuses were completed with both mouse and rat models. Quantification of novel parameters of digital images of thymuses from human infants suffering mortal head trauma revealed a disruption of the corticomedullary organization of the thymus, particularly involving dissolution of the corticomedullary border. A similar result was obtained for related mouse and rat models. The human thymuses from head trauma cases also displayed a higher percentage of Ki-67-positive thymocytes. Finally, we determined that thymus expression of the human serotonin receptor, and the serotonin transporter, occur almost exclusively in the thymic medulla. Head trauma leads to a disruption of the thymic, corticomedullary border, and molecular expression patterns in a robust and quantifiable manner.

Magnetic micelles for DNA delivery to rat brains after mild traumatic brain injury.

Traumatic brain injury (TBI) causes significant mortality, long term disability and psychological symptoms. Gene therapy is a promising approach for treatment of different pathological conditions. Here we tested chitosan and polyethyleneimine (PEI)-coated magnetic micelles (CP-mag micelles or CPMMs), a potential MRI contrast agent, to deliver a reporter DNA to the brain after mild TBI (mTBI). CPMM-tomato plasmid (ptd) conjugate expressing a red-fluorescent protein (RFP) was administered intranasally immediately after mTBI or sham surgery in male SD rats. Evans blue extravasation following mTBI suggested CPMM-ptd entry into the brain via the compromised blood-brain barrier. Magnetofection increased the concentration of CPMMs in the brain. RFP expression was observed in the brain (cortex and hippocampus), lung and liver 48 h after mTBI. CPMM did not evoke any inflammatory response by themselves and were excreted from the body. These results indicate the possibility of using intranasally administered CPMM as a theranostic vehicle for mTBI. From the clinical editor: In this study, chitosan and PEI-coated magnetic micelles (CPMM) were demonstrated as potentially useful vehicles in traumatic brain injury in a rodent model. Magnetofection increased the concentration of CPMMs in the brain and, after intranasal delivery, CPMM did not evoke any inflammatory response and were excreted from the body.

Multifunctional Chitosan Magnetic-Graphene (CMG) Nanoparticles: a Theranostic Platform for Tumor-targeted Co-delivery of Drugs, Genes and MRI Contrast Agents.

Combing chemotherapy with gene therapy has been one of the most promising strategies for the treatment of cancer. The noninvasive MRI with superparamagnetic iron oxide (SPIO) as contrast agent is one of the most effecitve techniques for evaluating the antitumor therapy. However, to construct a single system that can deliver efficiently gene, drug and SPIO to the cancer site remains a challenge. Herein, we report a chitosan functionalized magnetic graphene nanoparticle (CMG) platform for simultaneous gene/drug and SPIO delivery to tumor. The phantom and ex vivo MRI images suggest CMG as a strong T2 contrast-enhancing agent. The CMGs are biocompatible as evaluated by the WST assay and predominantly accumulate in tumors as shown by biodistribution studies and MRI. The anticancer drug doxorubicin (DOX) loaded CMGs (DOX-CMGs) release DOX faster at pH 5.1 than at pH 7.4, and more effective (IC50 = 2 µM) in killing A549 lung cancer cells than free DOX (IC50 = 4 µM). CMGs efficiently deliver DNA into A549 lung cancer cells and C42b prostate cancer cells. In addition, i.v. administration of GFP-plasmid encapsulated within DOX-CMGs into tumor-bearing mice has showed both GFP expression and DOX accumulation at the tumor site at 24 and 48 hrs after administration. These results indicate CMGs provide a robust and safe theranostic platform, which integrates targeted delivery of both gene medicine and chemotherapeutic drug(s), and enhanced MR imaging of tumors. The integrated chemo- and gene- therapeutic and diagnostic design of CMG nanoparticles shows promise for simultaneous targeted imaging, drug delivery and real -time monitoring of therapeutic effect for cancer.

Phospholipid micelle encapsulated gadolinium oxide nanoparticles for imaging and gene delivery.

We encapsulated gadolinium oxide (Gd2O3) nanoparticles within phospholipid micelles as a novel low cytotoxic T1-weighted MRI imaging contrast agent (MGdNPs) that can also deliver small molecules such as DNA plasmids. MGdNPs show relatively good MRI relaxivity values, negligible cytotoxicity, excellent cellular uptake and expression of DNA plasmids in vivo. Biodistribution studies in mice show that intranasal and intraperitoneal administration of MGdNPs can effectively target specific organs.

New perspectives on central and peripheral immune responses to acute traumatic brain injury.

Traumatic injury to the brain (TBI) results in a complex set of responses involving various symptoms and long-term consequences. TBI of any form can cause cognitive, behavioral and immunologic changes in later life, which underscores the problem of underdiagnosis of mild TBI that can cause long-term neurological deficits. TBI disrupts the blood-brain barrier (BBB) leading to infiltration of immune cells into the brain and subsequent inflammation and neurodegeneration. TBI-induced peripheral immune responses can also result in multiorgan damage. Despite worldwide research efforts, the methods of diagnosis, monitoring and treatment for TBI are still relatively ineffective. In this review, we delve into the mechanism of how TBI-induced central and peripheral immune responses affect the disease outcome and discuss recent developments in the continuing effort to combat the consequences of TBI and new ways to enhance repair of the damaged brain.

CCL20 Is Associated with Neurodegeneration Following Experimental Traumatic Brain Injury and Promotes Cellular Toxicity In Vitro.

Traumatic brain injury (TBI) is complex and involves multiple processes that contribute to functional decline. Progressive neuropathies result from delayed cellular death following the initial impact. Although the precise mechanisms responsible for delayed injury are unknown, numerous data implicate a role for the peripheral immune system in perpetuating neuroinflammation after TBI. A previous report demonstrated that splenic CCL20 chemokine expression was upregulated 24 h after lateral fluid percussive impact (LFPI), prior to neuronal expression but consistent with neurodegeneration. Here, we expand on those data to report increased CCL20 protein expression in white matter 48 h after LFPI and demonstrate that CCL20 is directly toxic to primary neurons and oligodendrocytes subjected to oxygen glucose deprivation. The temporal expression profile of CCL20, coupled with in vitro toxicity to primary cells, suggests that this chemokine exerts deleterious effects on cell viability following TBI. These findings warrant further investigations into the use of CCL20 as a potential biomarker and/or therapeutic target.

Lateral fluid percussion injury of the brain induces CCL20 inflammatory chemokine expression in rats.

BACKGROUND: Traumatic brain injury (TBI) evokes a systemic immune response including leukocyte migration into the brain and release of pro-inflammatory cytokines; however, the mechanisms underlying TBI pathogenesis and protection are poorly understood. Due to the high incidence of head trauma in the sports field, battlefield and automobile accidents identification of the molecular signals involved in TBI progression is critical for the development of novel therapeutics. METHODS: In this report, we used a rat lateral fluid percussion impact (LFPI) model of TBI to characterize neurodegeneration, apoptosis and alterations in pro-inflammatory mediators at two time points within the secondary injury phase. Brain histopathology was evaluated by fluoro-jade (FJ) staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, polymerase chain reaction (qRT PCR), enzyme linked immunosorbent assay (ELISA) and immunohistochemistry were employed to evaluate the CCL20 gene expression in different tissues. RESULTS: Histological analysis of neurodegeneration by FJ staining showed mild injury in the cerebral cortex, hippocampus and thalamus. TUNEL staining confirmed the presence of apoptotic cells and CD11b+ microglia indicated initiation of an inflammatory reaction leading to secondary damage in these areas. Analysis of spleen mRNA by PCR microarray of an inflammation panel led to the identification of CCL20 as an important pro-inflammatory signal upregulated 24 h after TBI. Although, CCL20 expression was observed in spleen and thymus after 24 h of TBI, it was not expressed in degenerating cortex or hippocampal neurons until 48 h after insult. Splenectomy partially but significantly decreased the CCL20 expression in brain tissues. CONCLUSION: These results demonstrate that the systemic inflammatory reaction to TBI starts earlier than the local brain response and suggest that spleen- and/ or thymus-derived CCL20 might play a role in promoting neuronal injury and central nervous system inflammation in response to mild TBI.

Immediate hypersensitivity to common food allergens: an investigation on food sensitization in respiratory allergic patients of calcutta, India.

BACKGROUND: : Food allergy may be defined as an immunoglobulin E-mediated immune response to food proteins. Such studies have previously not been done in Calcutta, India. The present study was therefore undertaken to record the sensitivity to commonly consumed foods in patients with allergic rhinitis and asthma. MATERIALS AND METHODS: : A survey of 800 patients (410 males and 390 females) reporting to the Allergy Unit of the Institute of Child Health, Calcutta, were selected for the study conducted from May 2006 to April 2007. Respiratory allergic patients in the age group of 5 to 60 years were evaluated using a standard questionnaire, and skin prick test was performed using common food and aeroallergens. RESULTS/CONCLUSIONS: : Out of the 684 patients with a history of food allergy, most of them, that is, 338, are in the age group 16 to 40 years, 192 of them were in the age group 41 to 60 years, and 154 were in the age group 5 to 15 years. Most of the patients with food allergy had asthma (65.05%), rhinitis and asthma (20.03%), and skin allergies (4.97%), such as itching, eczema, and urticaria. The foodstuffs that were found to elicit symptoms of hypersensitivity were egg, milk, wheat, pulses, vegetables, fishes, and fruits.The patients aged between 16 and 40 years (male-female ratio, 1:1.19) were mostly sensitive to prawn, brinjal, banana, ladyfinger, papaya, wheat, and egg. The age group 41 to 60 years (male-female ratio, 1:1.04) had high skin reactivity to brinjal, egg, banana, fish, and Phaseolus mungo. Patients younger than 16 years (male-female ratio, 1:1.33) were sensitized to brinjal, prawn, banana, spinach, and egg. We observed that food hypersensitivity also reflects different genetic factors and variations in cultural and dietary habits of each individual.

Microinfusion of pituitary adenylate cyclase-activating polypeptide into the central nucleus of amygdala of the rat produces a shift from an active to passive mode of coping in the shock-probe fear/defensive burying test.

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) nerve fibers are present in the central nucleus of amygdala (CeA), a brain region implicated in the control of fear-related behavior. This study evaluated PACAPergic modulation of fear responses at the CeA in male Sprague-Dawley rats. PACAP (50-100 pmol) microinfusion via intra-CeA cannulae produced increases in immobility and time the rats spent withdrawn into a corner opposite to the electrified probe compared to controls in the shock-probe fear/defensive burying test. Shock-probe burying and exploration, numbers of shocks received, locomotion distance, and velocity were all reduced by intra-CeA PACAP injection. Further, intra-CeA PACAP effects were manifested only when the animals were challenged by shock, as intra-CeA PACAP injections did not cause significant changes in the behaviors of unshocked rats. Thus, intra-CeA administration of PACAP produces a distinct reorganization of stress-coping behaviors from active (burying) to passive modes, such as withdrawal and immobility. These findings are potentially significant toward enhancing our understanding of the involvement of PACAP and the CeA in the neural basis of fear and anxiety.

Hypothalamic and brainstem sources of pituitary adenylate cyclase-activating polypeptide nerve fibers innervating the hypothalamic paraventricular nucleus in the rat.

The hypothalamic paraventricular nucleus (PVN) coordinates major neuroendocrine and behavioral mechanisms, particularly responses to homeostatic challenges. Parvocellular and magnocellular PVN neurons are richly innervated by pituitary adenylate cyclase-activating polypeptide (PACAP) axons. Our recent functional observations have also suggested that PACAP may be an excitatory neuropeptide at the level of the PVN. Nevertheless, the exact localization of PACAP-producing neurons that project to the PVN is not understood. The present study examined the specific contribution of various brain areas sending PACAP innervation to the rat PVN by using iontophoretic microinjections of the retrograde neuroanatomical tracer cholera toxin B subunit (CTb). Retrograde transport was evaluated from hypothalamic and brainstem sections by using multiple labeling immunofluorescence for CTb and PACAP. PACAP-containing cell groups were found to be retrogradely labeled from the PVN in the median preoptic nucleus; preoptic and lateral hypothalamic areas; arcuate, dorsomedial, ventromedial, and supramammillary nuclei; ventrolateral midbrain periaqueductal gray; rostral and midlevel ventrolateral medulla, including the C1 catecholamine cell group; nucleus of the solitary tract; and dorsal motor nucleus of vagus. Minor PACAP projections with scattered double-labeled neurons originated from the parabrachial nucleus, pericoeruleus area, and caudal regions of the nucleus of the solitary tract and ventrolateral medulla. These observations indicate a multisite origin of PACAP innervation to the PVN and provide a strong chemical neuroanatomical foundation for interaction between PACAP and its potential target neurons in the PVN, such as parvocellular CRH neurons, controlling physiologic responses to stressful challenges and other neuroendocrine or preautonomic PVN neurons.