A functional ultrasound brain GPS for automatic vascular-based neuronavigation.

Recent advances in ultrasound imaging triggered by transmission of ultrafast plane waves have rendered functional ultrasound (fUS) imaging a valuable neuroimaging modality capable of mapping cerebral vascular networks, but also for the indirect capture of neuronal activity with high sensitivity thanks to the neurovascular coupling. However, the expansion of fUS imaging is still limited by the difficulty to identify cerebral structures during experiments based solely on the Doppler images and the shape of the vessels. In order to tackle this challenge, this study introduces the vascular brain positioning system (BPS), a GPS of the brain. The BPS is a whole-brain neuronavigation system based on the on-the-fly automatic alignment of ultrafast ultrasensitive transcranial Power Doppler volumic images to common templates such as the Allen Mouse Brain Common Coordinates Framework. This method relies on the online registration of the complex cerebral vascular fingerprint of the studied animal to a pre-aligned reference vascular atlas, thus allowing rapid matching and identification of brain structures. We quantified the accuracy of the automatic registration using super-resolution vascular images obtained at the microscopic scale using Ultrasound Localization Microscopy and found a positioning error of 44 µm and 96 µm for intra-animal and inter-animal vascular registration, respectively. The proposed BPS approach outperforms the manual vascular landmark recognition performed by expert neuroscientists (inter-annotator errors of 215 µm and 259 µm). Using the online BPS approach coupled with the Allen Atlas, we demonstrated the capability of the system to position itself automatically over chosen anatomical structures and to obtain corresponding functional activation maps even in complex oblique planes. Finally, we show that the system can be used to acquire and estimate functional connectivity matrices automatically. The proposed functional ultrasound on-the-fly neuronavigation approach allows automatic brain navigation and could become a key asset to ensure standardized experiments and protocols for non-expert and expert researchers.

Differential regulation of NGF receptors in primary sensory neurons by adjuvant-induced arthritis in the rat.

In the adult brain, neurotrophins play a key role in adaptive processes linked to increased neuronal activity. A growing body of evidence suggests that chronic pain results from long-term plasticity of central pathways involved in nociception. We have investigated the involvement of nerve growth factor (NGF) in adaptive responses of primary sensory neurons during the course of a long-lasting inflammatory pain model. The amount and distribution of the NGF receptors p75(NTR) and TrkA were measured in the dorsal horn and dorsal root ganglia (DRG) of animals subjected to Freund's adjuvant-induced arthritis (AIA). We observed an increased immunoreactivity of both receptors in the central terminals of primary sensory neurons in the arthritic state. The increases were seen in the same population of afferent terminals in deep dorsal horn laminae. These changes paralleled the variations of clinical and behavioral parameters that characterize the course of the disease. They occurred in NGF-sensitive, but not GDNF-sensitive, nerve terminals. However, p75(NTR) and TrkA protein levels in the DRG (in the cell body of these neurons) showed different response patterns. An immediate rise of p75(NTR) was seen in parallel with the initial inflammation that developed after administration of Freund's adjuvant in hindpaws. In contrast, increases of the mature (gp140(trk)) form of TrkA occurred later and seemed to be linked to the development of the long-lasting inflammatory response. The changes in receptor expression were observed exclusively at lumbar levels, L3-L5, somatotopically appropriate for the inflammation. Together, these results implicate NGF in long-term mechanisms accompanying chronic inflammatory pain, via the up-regulation of its high affinity receptor, and offer additional evidence for differential processes underlying short- versus long-lasting inflammatory pain.

Chronic pain is associated with increased TrkA immunoreactivity in spinoreticular neurons.

Repetitive noxious stimulation leads to permanent adaptive changes of central pathways involved in the genesis and integration of nociception. Several classes of neurotrophic factors that affect brain plasticity are also involved in the regulation of sensory functions in adulthood. To investigate a putative role of nerve growth factor (NGF) in central plasticity linked to chronic pain, modifications in immunoreactivity (IR) for the high-affinity NGF receptor, TrkA, were studied at spinal levels in a rat model of inflammatory chronic pain, adjuvant-induced arthritis (AIA). We report a specific increase in the number of TrkA-IR profiles in laminae V-VI at lumbar levels L3 and L4 in arthritic rats. Tract tracing using FluoroGold injections in the ventrobasal complex of the thalamus and in the brainstem showed that these increased TrkA-IR profiles are spinoreticular neurons. Dual labeling with calcitonin gene-related peptide or substance P showed that TrkA-IR neurons were mainly located in projection fields of small- to medium-sized primary afferent fibers, which convey nociceptive inputs. These results suggest that TrkA-containing neurons of the spinal dorsal horn participate in the first central relay of transmission of nociceptive information to supraspinal centers. Enhanced numbers of TrkA-IR neurons during AIA strongly support the hypothesis of a participation of NGF in adaptive mechanisms of central nociceptive pathways observed in chronic pain states.

Matrix metalloproteinase gelatinases in sulfur mustard-induced acute airway injury in guinea pigs.

Respiratory tract lesions induced by sulfur mustard (SM), a chemical warfare agent, are characterized by epithelial damage associated with inflammatory cell infiltration. To test the potential role of matrix metalloproteinase gelatinases in these lesions, we evaluated gelatinase activity, albumin content, and total cell count in bronchoalveolar lavage fluid of guinea pigs 24 h after an intratracheal injection of 0.2 mg/kg of SM. The bronchial lavage and alveolar lavage fluids were analyzed separately. The increase in inflammatory cell content of the bronchial lavage fluid, mainly macrophages, observed in SM-intoxicated guinea pigs was accompanied by an increase in albumin and in 92-kDa gelatinase activity. There was a significant correlation between albumin content and 92-kDa gelatinase activity (r = 0.67) and between 92-kDa gelatinase and the number of macrophages. Immunohistochemistry performed on tracheal sections showed the presence of 92-kDa gelatinase at the site of intraepithelial cleavages. Zymography analysis of culture medium conditioned by guinea pig tracheal epithelial cells demonstrated that these cells produced in vitro 92-kDa gelatinase on stimulation. Culture of human bronchial epithelial cells obtained by the explant technique showed a marked increase in 92-kDa gelatinase after exposure to 5 x 10(-5) M SM that reinforced the relevance of our animal results to human exposure to SM. These results suggest that in SM respiratory intoxication, 92-kDa gelatinase of both inflammatory and epithelial cell origins could be involved in epithelial cell detachment.

Relationship of serum neutral endopeptidase E.C.3.4.24.11 activity to alcohol consumption.

Neutral endopeptidase (NEP) is an ubiquitous enzyme that inactivates numerous endogenous peptides in the brain, kidney, and lung in vivo. The origin of the circulating soluble form and its determinants are largely unknown. The relationships of smoking habits and alcohol consumption to serum NEP activity have been explored in a longitudinal epidemiological study conducted in 204 Lorraine coal miners. At both surveys, 4 years apart, NEP activity was significantly related to alcohol consumption (in grams/day) reported by questionnaire, with correlation coefficients of 0.26 (p = 0.001) in 1990 and 0.23 (p = 0.001) in 1994. The correlations of gamma-glutamyltransferase to NEP activity were even stronger with correlation coefficients of 0.71 (p = 0.0001) in 1990 and 0.79 (p = 0.0001) in 1994. Longitudinally, the change in NEP activity between the first and the second surveys was significantly correlated with change in alcohol consumption (r = to 0.18, p = 0.02) and with change in gamma-glutamyltransferase level (r = 0.60, p = 0.0001). Serum NEP activity was unrelated to smoking habits. Results support the hypothesis of a causal role of alcohol on serum NEP activity.

Oxidant-antioxidant balance in alveolar macrophages from newborn rats.

An oxidant-antioxidant imbalance in neonatal alveolar macrophages (AMs) may contribute to the increased susceptibility to lung injury described in the neonatal period. We therefore evaluated oxygen radical production by rat AMs at various postnatal ages, and measured in parallel cellular antioxidant enzyme activities. AMs were obtained by bronchoalveolar lavage from rats aged < 24 h, 21 days and 5 weeks, and results were compared to those obtained with adult rat AMs. Intracellular production of oxygen radical species, estimated fluorometrically using 2',5'-dichlorofluorescein diacetate as the substrate, was significantly reduced in neonates as compared with adults, both in the presence and in the absence of cell stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. A similar pattern was observed for the extracellular release of oxygen radical species, evaluated by lucigenin-enhanced chemiluminescence (CL) or peroxidase-catalysed CL oxidation of luminol: peak CL values measured after cell stimulation with PMA or opsonized zymosan remained significantly lower for AMs from newborn rats than for AMs from adults. By contrast, high values for antioxidant enzyme activities (superoxide dismutase and glutathione peroxidase) in AMs were demonstrated in newborns as compared to adults. We conclude that high antioxidant activity in rat AMs after birth may be at least partly responsible for the low production of oxygen metabolites observed during the same period.

Tachykinins induce gelatinase production by guinea pig alveolar macrophages: involvement of NK2 receptors.

To determine whether tachykinins induce gelatinase production by guinea pig alveolar macrophages (AM), and to characterize the mechanism involved, we incubated AM with substance P (SP), neurokinin A (NKA), or the NH2-terminal fragment of SP, SP(1-7). The effects of increasing concentrations of selective NK1 and NK2 agonists on tachykinin-induced gelatinase production were also evaluated, as were the effects of a selective NK2 antagonist. Gelatinase activity in conditioned culture media (CCM) was assessed by zymography and quantified by image analysis. SP increased 92-kDa gelatinase activity in CCM of AM in a concentration-dependent manner, with a maximum increase at 10(-4) M. NKA, the NH2-terminal fragment of SP, and an NK1-selective agonist had no effect. In contrast, a selective NK2 agonist induced a concentration-dependent increase in gelatinase activity. The increase in this activity induced by SP and the selective NK2 agonist was inhibited by a selective NK2 antagonist. We conclude that SP induces gelatinase production by AM through NK2 receptor activation. The release of gelatinase may constitute one mechanism through which SP contributes to the epithelial lesions observed in bronchial hyperreactivity and asthma.

Increased 92 kD gelatinase activity from alveolar macrophages in newborn rats.

The functional immaturity of neonatal alveolar macrophages (AM) may contribute to the increased susceptibility of neonates to lung injury. Because the secretion of proteinases by neonatal AMs may be involved in normal postnatal lung development and in repair after lung injury, we evaluated the capacity of neonatal AMs to secrete 92 kD Type IV collagenase. AMs were obtained by bronchoalveolar lavage from newborn rats at different postnatal ages. Total gelatinase activity was measured by zymography in AM-conditioned media. Spontaneous secretion of gelatinase from AMs varied significantly with age, the highest levels being found immediately after birth. Stimulation of AMs by PMA induced a four- to fivefold greater increase in total gelatinase activity during the first 10 d of postnatal life compared with adulthood. Using [3H]gelatin as the substrate, we found high free gelatinase activity only within 24 h after birth; data obtained after exposing cells to natural surfactant suggested that surfactant may account in part for this increase in free gelatinase activity. No secretion of tissue inhibitor of metalloproteinases (TIMP) by AMs was detectable in newborns within 24 h after birth. We conclude that AMs from newborn rats are able to secrete more gelatinase than AMs from adults, and this enzyme production profile during the neonatal period may contribute to the fact that newborns with lung injury are at high risk for extracellular matrix degradation.

Matrix metalloproteinase and elastase activities in LPS-induced acute lung injury in guinea pigs.

Matrix metalloproteinases (MMPs) and elastase are proteolytic enzymes specifically directed against extracellular matrix (ECM) components. They are secreted by inflammatory cells and may consequently contribute to the lesions of the ECM observed during acute pulmonary edema. We therefore evaluated the MMP and elastase activities, which are secreted by cultured alveolar macrophages (AMACs) and polymorphonuclear neutrophils (PMNs) and present in the bronchoalveolar lavage (BAL) fluid in a guinea pig model of acute lung injury induced by intratracheal instillation of lipopolysaccharide (LPS). The control group was given 0.9% NaCl. 24 h after instillation, a BAL was performed, the BAL fluid was separated from the cells by centrifugation, and AMACs and PMNs were separately cultured for 24 h. In BAL fluid from LPS-treated guinea pigs, we found 1) an increase in free gelatinase activity, tested on [3H]gelatin (0.7 +/- 0.2 micrograms.200 microliters BAL fluid-1.48 h-1 vs. 0.2 +/- 0.1 in controls, P < 0.05), and 2) increased total gelatinase activities, as assessed by zymography. The molecular masses of the major gelatinase species found in BAL fluid by zymography were 92 and 68 kDa. The 92-kDa gelatinase was secreted by both AMACs and PMNs, as demonstrated by zymography of their respective culture media. When tested on [3H]elastin, the elastase activity of BAL fluid of LPS-treated animals exhibited no increase, but when tested on a synthetic peptidic substrate [N-succinyl-(L-alanine)3-p-nitro anilide (SLAPN)], increased elastase-like activity was observed (from 17 +/- 4 nmol of SLAPN.200 microliters BAL fluid-1.24 h-1 in control group to 34 +/- 8 in LPS group, P < 0.05). This increase was attributable to the activity of a metalloendopeptidase that was inhibited by the metal chelator EDTA but not by the specific tissue inhibitor of MMPs.