Absence of functional deficits in rats following systemic administration of an AAV9 vector despite moderate peripheral nerve and dorsal root ganglia findings: A clinically silent peripheral neuropathy.

Adeno-associated virus (AAV)-based vectors are commonly used for delivering transgenes in gene therapy studies, but they are also known to cause dorsal root ganglia (DRG) and peripheral nerve toxicities in animals. However, the functional implications of these pathologic findings and their time course remain unclear. At 2, 4, 6, and 8 weeks following a single dose of an AAV9 vector carrying human frataxin transgene in rats, non-standard functional assessments, including von Frey filament, electrophysiology, and Rotarod tests, were conducted longitudinally to measure allodynia, nerve conduction velocity, and coordination, respectively. Additionally, DRGs, peripheral nerves, brain and spinal cord were evaluated histologically and circulating neurofilament light chain (NfL) was quantified at 1, 2, 4, and 8 weeks, respectively. At 2 and 4 weeks after dosing, minimal-to-moderate nerve fiber degeneration and neuronal degeneration were observed in the DRGs in some of the AAV9 vector-dosed animals. At 8 weeks, nerve fiber degeneration was observed in DRGs, with or without neuronal degeneration, and in sciatic nerves of all AAV9 vector-dosed animals. NfL values were higher in AAV9 vector-treated animals at weeks 4 and 8 compared with controls. However, there were no significant differences in the three functional endpoints evaluated between the AAV9 vector- and vehicle-dosed animals, or in a longitudinal comparison between baseline (predose), 4, and 8 week values in the AAV9 vector-dose animals. These findings demonstrate that there is no detectable functional consequence to the minimal-to-moderate neurodegeneration observed with our AAV9 vector treatment in rats, suggesting a functional tolerance or reserve for loss of DRG neurons after systemic administration of AAV9 vector.

Methodologies and Emerging Technologies for the Evaluation of the Hematopoietic System.

Hematology and bone marrow analysis is central to our understanding of the hematopoietic system and how it responds to insults, and this session presented during the 2022 STP symposium provided a review of current and novel approaches for the evaluation of the hematopoietic system in the context of nonclinical investigations. This publication summarizes the information presented on novel approaches for evaluation of the hematopoietic system using automated hematology analyzers, including details around the quantitative assessment of bone marrow cell suspensions as well as introducing several newly available hematology parameters. It was followed by a discussion on intravital microscopy and live cell imaging and how these methods can assist with de-risking hematopoiesis-associated safety concerns, and a review of recent assays using artificial intelligence for the evaluation of bone marrow.

Comprehensive Nonclinical Safety Assessment of Nirmatrelvir Supporting Timely Development of the SARS-COV-2 Antiviral Therapeutic, Paxlovid™.

COVID-19 is a potentially fatal infection caused by the SARS-CoV-2 virus. The SARS-CoV-2 3CL protease (Mpro) is a viral enzyme essential for replication and is the target for nirmatrelvir. Paxlovid (nirmatrelvir co-administered with the pharmacokinetic enhancer ritonavir) showed efficacy in COVID-19 patients at high risk of progressing to hospitalization and/or death. Nonclinical safety studies with nirmatrelvir are essential in informing benefit-risk of Paxlovid and were conducted to support clinical development. In vivo safety pharmacology assessments included a nervous system/pulmonary study in rats and a cardiovascular study in telemetered monkeys. Potential toxicities were assessed in repeat dose studies of up to 1 month in rats and monkeys. Nirmatrelvir administration (1,000 mg/kg, p.o.) to male rats produced transient increases in locomotor activity and respiratory rate but did not affect behavioral endpoints in the functional observational battery. Cardiovascular effects in monkeys were limited to transient increases in blood pressure and decreases in heart rate, observed only at the highest dose tested (75 mg/kg per dose b.i.d; p.o.). Nirmatrelvir did not prolong QTc-interval or induce arrhythmias. There were no adverse findings in repeat dose toxicity studies up to 1 month in rats (up to 1,000 mg/kg daily, p.o.) or monkeys (up to 600 mg/kg daily, p.o.). Nonadverse, reversible clinical pathology findings without clinical or microscopic correlates included prolonged coagulation times at ≥60 mg/kg in rats and increases in transaminases at 600 mg/kg in monkeys. The safety pharmacology and nonclinical toxicity profiles of nirmatrelvir support clinical development and use of Paxlovid for treatment of COVID-19.

Circulating neurofilament light chain as a promising biomarker of AAV-induced dorsal root ganglia toxicity in nonclinical toxicology species.

Adeno-associated virus (AAV)-induced dorsal root ganglia (DRG) toxicity has been observed in several nonclinical species, where lesions are characterized by neuronal degeneration/necrosis, nerve fiber degeneration, and mononuclear cell infiltration. As AAV vectors become an increasingly common platform for novel therapeutics, non-invasive biomarkers are needed to better characterize and manage the risk of DRG neurotoxicity in both nonclinical and clinical studies. Based on biological relevance, reagent availability, antibody cross-reactivity, DRG protein expression, and assay performance, neurofilament light chain (NF-L) emerged as a promising biomarker candidate. Dose- and time-dependent changes in NF-L were evaluated in male Wistar Han rats and cynomolgus monkeys following intravenous or intrathecal AAV injection, respectively. NF-L profiles were then compared against microscopic DRG lesions on day 29 post-dosing. In animals exhibiting DRG toxicity, plasma/serum NF-L was strongly associated with the severity of neuronal degeneration/necrosis and nerve fiber degeneration, with elevations beginning as early as day 8 in rats (≥5 × 1013 vg/kg) and day 14 in monkeys (≥3.3 × 1013 vg/dose). Consistent with the unique positioning of DRGs outside the blood-brain barrier, NF-L in cerebrospinal fluid was only weakly associated with DRG findings. In summary, circulating NF-L is a promising biomarker of AAV-induced DRG toxicity in nonclinical species.

Biodistribution and Tolerability of AAV-PHP.B-CBh-SMN1 in Wistar Han Rats and Cynomolgus Macaques Reveal Different Toxicologic Profiles.

Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high-dose intravenous (IV) administration. To characterize biodistribution, transduction, and toxicity among common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron 1 (SMN1) transgene (AAV-PHP.B-CBh-SMN1) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2 × 1013, 5 × 1013, or 1 × 1014 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5 × 1013 vg/kg was found dead on day 4. Clinical pathology data on days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi, suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while quantitative polymerase chain reaction confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after 1 month in rats, and suggest adding the autonomic system in the watch list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.

Evaluation of the Central Effects of Systemic Lentiviral-Mediated Leptin Delivery in Streptozotocin-Induced Diabetic Rats.

Type 1 diabetes (T1D) is characterized by hyperphagia, hyperglycemia and activation of the hypothalamic-pituitary-adrenal (HPA) axis. We have reported previously that daily leptin injections help to alleviate these symptoms. Therefore, we hypothesized that leptin gene therapy could help to normalize the neuroendocrine dysfunction seen in T1D. Adult male Sprague Dawley rats were injected i.v. with a lentiviral vector containing the leptin gene or green fluorescent protein. Ten days later, they were injected with the vehicle or streptozotocin (STZ). HPA function was assessed by measuring norepinephrine (NE) levels in the paraventricular nucleus (PVN) and serum corticosterone (CS). Treatment with the leptin lentiviral vector (Lepvv) increased leptin and insulin levels in non-diabetic rats, but not in diabetic animals. There was a significant reduction in blood glucose levels in diabetic rats due to Lepvv treatment. Both NE levels in the PVN and serum CS were reduced in diabetic rats treated with Lepvv. Results from this study provide evidence that leptin gene therapy in STZ-induced diabetic rats was able to partially normalize some of the neuroendocrine abnormalities, but studies with higher doses of the Lepvv are needed to develop this into a viable option for treating T1D.

Responsiveness of hypothalamo-pituitary-adrenal axis to leptin is impaired in diet-induced obese rats.

BACKGROUND/OBJECTIVES: Diet-induced obese (DIO) rats have altered stress (HPA) axis activity compared to diet-resistant (DR) rats when chronically exposed to a high-fat (HF) diet. Since stress axis is tightly regulated by leptin, an adipocyte-secreted hormone that is important for controlling body weight, we hypothesized that leptin action is impaired in DIO rats leading to alterations in HPA axis activity. SUBJECTS/METHODS: We intraperitoneally injected selectively bred DIO and DR rats with either saline or recombinant rat leptin. HPA axis activity was assessed by measuring norepinephrine (NE) in the paraventricular nucleus (PVN), corticotropin-releasing hormone (CRH) in the median eminence, and serum corticosterone (CORT). To test if HF exposure duration and the corresponding increase in leptin differentially affects HPA axis activity, we placed animals on a chow or HF diet for 1 or 6 weeks. RESULTS: Leptin injection significantly increased serum leptin levels in both DIO and DR animals. It also reduced PVN NE in both groups, indicating that noradrenergic neurons in both groups remain responsive to leptin. HF diet duration-dependently increased serum leptin only in DIO animals whereas PVN NE increased in both groups. While DR rats responded to HF diet by increasing CRH and CORT at both time-points, responses in DIO rats varied, suggesting that they have altered HPA axis activity that may be dependent on HF-induced leptin levels and/or signaling. To understand the underlying mechanisms, we measured pSTAT-3, a marker of leptin signaling, in brainstem noradrenergic neurons and found reduced pSTAT-3 in A1 region of HF-fed DIO rats. We also found higher serum free fatty acids (FFAs) and a pro-inflammatory cytokine, IL-1ß. CONCLUSIONS: Collectively, these findings reveal that DIO rats have inherent neuroendocrine impairment in NE-HPA axis circuitry that worsens with the extent of HF diet exposure, possibly due to brainstem leptin resistance and/or elevated circulating FFAs and IL-1ß.

Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis.

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo(-/-)) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

Increased extracellular pressure stimulates tumor proliferation by a mechanosensitive calcium channel and PKC-ß.

Large tumors exhibit high interstitial pressure heightened by growth against the constraining stroma. Such pressures could stimulate tumor proliferation via a mechanosensitive ion channel. We studied the effects of 0-80 mmHg increased extracellular pressure for 24 h on proliferation of SW620, Caco-2, and CT-26 colon; MCF-7 breast; and MLL and PC3 prostate cancer cells, and delineated its mechanism in SW620 cells with specific inhibitors and siRNA. Finally, we compared NF-kB, phospho-IkB and cyclin D1 immunoreactivity in the high pressure centers and low pressure peripheries of human tumors. Pressure-stimulated proliferation in all cells. Pressure-driven SW620 proliferation required calcium influx via the T-type Ca(2+) channel Cav3.3, which stimulated PKC-ß to invoke the IKK-IkB-NF-kB pathway to increase proliferation and S-phase fraction. The mitotic index and immunoreactivity of NF-kB, phospho-IkB, and cyclin D1 in the center of 28 large human colon, lung, and head and neck tumors exceeded that in tumor peripheries. Extracellular pressure increases [Ca(2+)]i via Cav3.3, driving a PKC-ß- IKK- IkB-NF-kB pathway that stimulates cancer cell proliferation. Rapid proliferation in large stiff tumors may increase intratumoral pressure, activating this pathway to stimulate further proliferation in a feedback cycle that potentiates tumor growth. Targeting this pathway may inhibit proliferation in large unresectable tumors.

Chronic estradiol-17ß exposure suppresses hypothalamic norepinephrine release and the steroid-induced luteinizing hormone surge: role of nitration of tyrosine hydroxylase.

Chronic exposure to estrogens is known to produce a variety of deleterious effects in women including breast and ovarian cancer and anovulation. In female rats, exposure to low levels of estradiol-17ß (E2) decreases hypothalamic norepinephrine (NE) to suppress luteinizing hormone (LH) secretion and cause failure of ovulation. We hypothesized that E2 exposure most likely decreases NE release in the medial preoptic area (MPA) of the hypothalamus to produce this effect and that this may be due to E2-induced inflammatory changes in noradrenergic nuclei leading to nitration of an enzyme involved in NE synthesis. To test this, female Sprague Dawley rats were sham implanted or implanted with slow release E2 pellets (20ng/day) for 30, 60 or 90 days (E30, E60 and E90 respectively). At the end of the treatment period, the rats were implanted with a push-pull cannula in the MPA, ovariectomized and steroid primied to induce a LH surge and subjected to push-pull perfusion. Perfusates were analyzed for NE levels using HPLC-EC. Blood samples collected simultaneously were analyzed for LH levels. We measured interleukin-1ß (IL-1ß) and nitrate levels in brainstem noradrenergic nuclei that innervate the MPA. In control animals, there was a marked increase in NE levels in response to steroid priming at 1600h that was reduced in the E30 group, and completely abolished after 60 and 90 days of E2 exposure. LH profiles were similar to NE release profiles in control and E2-treated animals. We found that IL-1ß levels increased in all three (A1, A2 and A6) noradrenergic nuclei with chronic E2 exposure, while nitrate levels increased only in the A6 region. There was an increase in the nitration of the NE synthesizing enzyme in the MPA in this group as well probably contributing to reduced NE synthesis. This could be a possible mechanism by which chronic E2 exposure decreases NE levels in the MPA to suppress the LH surge.

Differential effects of inhalation exposure to PM2.5 on hypothalamic monoamines and corticotrophin releasing hormone in lean and obese rats.

Acute exposure to airborne pollutants, especially particulate matter (PM2.5) is known to increase hospital admissions for cardiovascular conditions, increase cardiovascular related mortality and predispose the elderly and obese individuals to cardiovascular conditions. The mechanisms by which PM2.5 exposure affects the cardiovascular system is not clear. Since the autonomic system plays an important role in cardiovascular regulation, we hypothesized that PM2.5 exposure most likely activates the paraventricular nucleus (PVN) of the hypothalamus to cause an increase in sympathetic nervous system and/or stress axis activity. We also hypothesized that these changes may be sustained in obese rats predisposing them to higher cardiovascular risk. To test this, adult male Brown Norway (BN) rats were subjected to one day or three days of inhalation exposures to filtered air (FA) or concentrated air particulate (CAP) derived from ambient PM2.5. Corpulent JCR-LA rats were exposed to FA or CAP for four days. Animals were sacrificed 24h after the last inhalation exposure. Their brains were removed, frozen and sectioned. The PVN and median eminence (ME) were microdissected. PVN was analyzed for norepinephrine (NE), dopamine (DA) and 5-hydroxy-indole acetic acid (5-HIAA) levels using HPLC-EC. ME was analyzed for corticotrophin releasing hormone (CRH) levels by ELISA. One day exposure to CAP increased NE levels in the PVN and CRH levels in the ME of BN rats. Repeated exposures to CAP did not affect NE levels in the PVN of BN rats, but increased NE levels in JCR/LA rats. A similar pattern was observed with 5-HIAA levels. DA levels on the other hand, were unaffected in both BN and JCR/LA strains. These data suggest that repeated exposures to PM2.5 continue to stimulate the PVN in obese animals but not lean rats.

Interleukin-1 beta simultaneously affects the stress and reproductive axes by modulating norepinephrine levels in different brain areas.

AIMS: Interleukin-1ß (IL-1ß) is a cytokine that is known to activate the stress axis and suppress the reproductive axis. Different brain areas are involved in the regulation of these two axes. However, they are both under the stimulatory control of the catecholamine, norepinephrine (NE). Here, we hypothesized that IL-1ß differentially affects these two axes by modulating NE levels in specific brain regions. MAIN METHODS: Female Sprague-Dawley rats in proestrus were injected intraperitoneally with either PBS-1.0% BSA (control) or 5µg of IL-1ß at 1pm. Groups of rats were sacrificed at 1, 3, and 5pm and their brains were collected. Brain areas associated with reproduction as well as areas associated with stress axis activity were isolated and analyzed for NE concentrations using HPLC-EC. Trunk blood was analyzed for IL-1ß, corticosterone and luteinizing hormone levels. KEY FINDINGS: As a general trend, treatment with IL-1ß significantly decreased NE levels (p<0.05) in the areas controlling reproductive functions when compared to the control group. In contrast, NE levels increased significantly (p<0.05) in the stress associated areas. LH levels were markedly decreased with IL-1ß treatment while corticosterone levels increased dramatically. SIGNIFICANCE: The ability of IL-1ß to produce differential effects on the stress and reproductive axis could be explained by modulation of NE levels in specific brain areas that are associated with these functions. This differential regulation of NE may be an adaptive phenomenon in response to a systemic immune challenge.

Differential effects of systemic interleukin-1ß on gene expression in brainstem noradrenergic nuclei.

AIMS: The cytokine, interleukin-1ß (IL-1ß), is known to produce specific effects on the neuroendocrine system such as suppression of the reproductive axis and stimulation of the stress axis. The mechanism by which IL-1ß produces these differential effects is not clear. Since norepinephrine (NE) is involved in these effects, we hypothesized that IL-1ß acts on brainstem noradrenergic nuclei to affect gene transcription of NE synthesizing enzymes, cytokines and associated transcription factors. MAIN METHODS: Adult female Sprague Dawley rats in proestrus were divided into two groups. Control animals received PBS-BSA and the treatment group received 5 µg of rat recombinant IL-1ß i.p. at noon. They were sacrificed in groups at 1, 3 and 5 pm (n=6/group) for measurement of tyrosine hydroxylase (TH) mRNA by qPCR or at 3 pm for mRNA analysis by qPCR array. KEY FINDINGS: TH mRNA levels decreased gradually with time in both control and IL-1ß-treated rats in the ventrolateral medulla. In the nucleus of solitary tract, TH mRNA levels were significantly reduced by IL-1ß treatment at 5 pm. In the locus coeruleus, TH mRNA levels increased significantly at 5 pm with IL-1ß treatment compared to controls. In the second set of animals analyzed by qPCR array, there were several fold increases in the expression of certain cytokines, chemokines, and transcription factors in specific noradrenergic nuclei. SIGNIFICANCE: Systemic administration of IL-1ß causes significant changes in the expression of tyrosine hydroxylase and several chemokines in brain stem noradrenergic nuclei, thereby mediating its neuroendocrine effects.

Interaction between GABA and norepinephrine in interleukin-1beta-induced suppression of the luteinizing hormone surge.

Interleukin-1beta (IL-1beta), a cytokine that is closely associated with inflammation and immune stress, is known to interfere with reproductive functions. Earlier studies have demonstrated that IL-1beta inhibits the luteinizing hormone (LH) surge during the afternoon of proestrus in female rats. We have shown that this effect is most probably mediated through a reduction in norepinephrine (NE) levels in the medial preoptic area (MPA) of the hypothalamus. However, the mechanism by which IL-1beta decreases NE levels in the MPA is unclear. We hypothesized that the inhibitory neurotransmitter, GABA could play a role in decreasing NE levels in the MPA. To test this, ovariectomized, steroid-primed rats were injected (i.p.) with either PBS-BSA (control) or 5 microg of IL-1beta, alone or in combination with i.c.v. administration of GABA-A and GABA-B receptor antagonists, Bicuculline and CGP 35348 (CGP) respectively. Animals were subjected to push-pull perfusion of the MPA and perfusates collected at 30 min intervals were analyzed for both NE and GABA levels using HPLC-EC. Simultaneously, serial plasma samples were obtained through jugular catheters and were analyzed for LH levels using RIA. Compared to control rats, NE levels decreased significantly in the MPA in IL-1beta-treated rats (p<0.05). Concurrently, there was a significant increase in GABA levels in the MPA (p<0.05). The GABA-A receptor antagonist, bicuculline, was able to reverse the effect of IL-1beta on NE and LH, while the GABA-B receptor antagonist, CGP 35348 was without any effect. This leads us to conclude that the IL-1beta-induced suppression of the LH surge is most probably mediated through an increase in GABA levels in the MPA which causes a reduction in NE levels. This is probably one of the mechanisms by which IL-1beta inhibits reproductive functions.

Systemic administration of leptin decreases plasma corticosterone levels: role of hypothalamic norepinephrine.

Leptin, an adipocyte-derived hormone, is known to regulate a variety of neuroendocrine functions. It inhibits the hypothalamo-pituitary-adrenal axis (HPA) in several animal models, however, the exact mechanism by which it does so is not known. Since norepinephrine (NE) is a key regulator of the HPA axis, we hypothesized that leptin could suppress HPA activity by decreasing NE levels. To study this, we implanted adult male Sprague-Dawley rats with both a push-pull cannula in the paraventricular nucleus (PVN) and a catheter in the jugular vein. Animals were treated with either 0 or 100 microg or 500 microg of recombinant rat leptin (Lep). Push-pull perfusion was performed from 1000-1600 h. Perfusate samples were collected every 30 min and analyzed for NE levels using HPLC-EC. Blood samples were collected every 60 min and analyzed for corticosterone (CS) levels. To further understand the role of NE in this phenomenon animals were treated with either an alpha1-adrenergic agonist, phenylephrine (PHE; 0.5 mg/kg BW), an alpha2-adrenergic agonist, clonidine (CLON; 0.6 mg/kg BW), or a beta-adrenergic agonist, isoproterenol (ISO; 0.2 mg/kg BW) alone or in combination with 500 microg of Lep. Pre-treatment and hourly post-treatment blood samples were collected, plasma was separated and analyzed for CS levels. Leptin administration decreased NE release in the PVN significantly by 30 min (p<0.05). It also significantly reduced plasma CS levels at 240 and 300 min (p<0.05). Administration of either PHE or CLON in combination with leptin prevented the leptin-induced decrease in CS. In contrast, administration of ISO along with leptin did not prevent the leptin-induced decrease in CS. These results indicate that leptin decreases hypothalamic NE and plasma CS and that this effect is most probably mediated through alpha-adrenergic receptors.

Developmental programming of cardiovascular disorders: focus on hypertension.

Increasing evidence suggests that adult cardiovascular disorders, e.g. hypertension, can be "programmed" in utero. The mechanisms that affect the developing fetus and lead to future cardiovascular disease are not fully established. This review addresses the possible involvement of maternal nutrition, sex steroids and other endocrine factors in the programming of hypertension in adulthood. Some possible mechanisms of subsequent development of hypertension in adulthood, such as elevated sympathetic and renin-angiotensin system activity, and failure of nephron development, also are discussed. Previous studies suggest that maternal undernutrition could be a major factor in fetal programming, but in light of the increased worldwide prevalence of obesity, maternal overnutrition is now receiving increased attention. Special emphasis is given here to this phenomenon. Obesity is associated with increased serum and tissue levels of proinflammatory cytokines, and loss of sensitivity to the adipokine leptin. It is postulated that this causes dysregulation of the hypothalamo-pituitary-adrenal axis, resulting in increased levels of circulating glucocorticoids. These factors could play a major role in programming, during the in utero period, of future hypertension in the offspring of obese mothers.

Activation of the stress axis and neurochemical alterations in specific brain areas by concentrated ambient particle exposure with concomitant allergic airway disease.

OBJECTIVE: Exposure to ambient particulate matter (PM) has been linked to respiratory diseases in people living in urban communities. The mechanism by which PM produces these diseases is not clear. We hypothesized that PM could act on the brain directly to stimulate the stress axis and predispose individuals to these diseases. The purpose of this study was to test if exposure to PM can affect brain areas involved in the regulation of neuroendocrine functions, especially the stress axis, and to study whether the presence of preexisting allergic airway disease aggravates the stress response. DESIGN: Adult male rats (n = 8/group) with or without ovalbumin (OVA)-induced allergic airway disease were exposed to concentrated air particles containing PM with an aerodynamic diameter pound 2.5 microm (PM(2.5)) for 8 hr, generated from ambient air in an urban Grand Rapids, Michigan, community using a mobile air research laboratory (AirCARE 1). Control animals were exposed to normal air and were treated with saline. MEASUREMENTS: A day after PM(2.5) exposure, animals were sacrificed and the brains were removed, frozen, and sectioned. The paraventricular nucleus (PVN) and other brain nuclei were microdissected, and the concentrations of aminergic neurotransmitters and their metabolites were measured using high-performance liquid chromatography with electrochemical detection. Serum corticosterone levels were measured using radioimmunoassay. RESULTS: A significant increase in the concentration (mean +/- SE, pg/microg protein) of norepinephrine in the PVN was produced by exposure to concentrated ambient particles (CAPs) or OVA alone (12.45 +/- 2.7 and 15.84 +/- 2.8, respectively) or after sensitization with OVA (19.06 +/- 3.8) compared with controls (7.98 +/- 1.3 ; p < 0.05). Serum corticosterone (mean +/- SE, ng/mL) was significantly elevated in the OVA + CAPs group (242.786 +/- 33.315) and in the OVA-presensitized group (242.786 +/- 33.315) compared with CAP exposure alone (114.55 +/- 20.9). Exposure to CAPs (alone or in combination with OVA pretreatment) can activate the stress axis, and this could probably play a role in aggravating allergic airway disease.