Vladimir A. Kulchitsky

Signaling the brain in systemic inflammation: which vagal branch is involved in fever genesis?

Recent evidence has suggested a role of abdominal vagal afferents in the pathogenesis of the febrile response. The abdominal vagus consists of five main branches (viz., the anterior and posterior celiac branches, anterior and posterior gastric branches, and hepatic branch). The branch responsible for transducing a pyrogenic signal from the periphery to the brain has not as yet been identified. In the present study, we address this issue by testing the febrile responsiveness of male Wistar rats subjected to one of four selective vagotomies: celiac (CBV), gastric (GBV), hepatic (HBV), or sham (SV). In the case of CBV, GBV, and HBV, only the particular vagal branch(es) was cut; for SV, all branches were left intact. After the postsurgical recovery (26-29 days), the rats had a catheter implanted into the jugular vein. On days 29-32, their colonic temperature (Tc) responses to a low dose (1 microg/kg) of Escherichia coli lipopolysaccharide (LPS) were studied. Three days later, the animals were subjected to a 24-h food and water deprivation, and the effectiveness of the four vagotomies to induce gastric food retention, pancreatic hypertrophy, and impairment of the portorenal osmotic reflex was assessed by weighing the stomach and pancreas and measuring the specific gravity of bladder urine, respectively. Stomach mass, pancreas mass, and urine density successfully separated the four experimental groups into four distinct clusters, thus confirming that each type of vagotomy had a different effect on the indexes measured. The Tc responses of SV, CBV, and GBV rats to LPS did not differ and were characterized by a latency of approximately 40 min and a maximal rise of 0.7 +/- 0.1, 0.6 +/- 0.1, and 0.9 +/- 0.2 degrees C, respectively. The fever response of the HBV rats was different; practically no Tc rise occurred (0.1 +/- 0.2 degrees C). The HBV appeared to be the only selective abdominal vagotomy affecting the febrile responsiveness. We conclude, therefore, that the hepatic vagus plays an important role in the transduction of a pyrogenic signal from the periphery to the brain.Recent evidence has suggested a role of abdominal vagal afferents in the pathogenesis of the febrile response. The abdominal vagus consists of five main branches (viz., the anterior and posterior celiac branches, anterior and posterior gastric branches, and hepatic branch). The branch responsible for transducing a pyrogenic signal from the periphery to the brain has not as yet been identified. In the present study, we address this issue by testing the febrile responsiveness of male Wistar rats subjected to one of four selective vagotomies: celiac (CBV), gastric (GBV), hepatic (HBV), or sham (SV). In the case of CBV, GBV, and HBV, only the particular vagal branch(es) was cut; for SV, all branches were left intact. After the postsurgical recovery (26-29 days), the rats had a catheter implanted into the jugular vein. On days 29-32, their colonic temperature (Tc) responses to a low dose (1 μg/kg) of Escherichia colilipopolysaccharide (LPS) were studied. Three days later, the animals were subjected to a 24-h food and water deprivation, and the effectiveness of the four vagotomies to induce gastric food retention, pancreatic hypertrophy, and impairment of the portorenal osmotic reflex was assessed by weighing the stomach and pancreas and measuring the specific gravity of bladder urine, respectively. Stomach mass, pancreas mass, and urine density successfully separated the four experimental groups into four distinct clusters, thus confirming that each type of vagotomy had a different effect on the indexes measured. The Tc responses of SV, CBV, and GBV rats to LPS did not differ and were characterized by a latency of ∼40 min and a maximal rise of 0.7 ± 0.1, 0.6 ± 0.1, and 0.9 ± 0.2°C, respectively. The fever response of the HBV rats was different; practically no Tc rise occurred (0.1 ± 0.2°C). The HBV appeared to be the only selective abdominal vagotomy affecting the febrile responsiveness. We conclude, therefore, that the hepatic vagus plays an important role in the transduction of a pyrogenic signal from the periphery to the brain.

Methodology of fever research: why are polyphasic fevers often thought to be biphasic?

This study explains why the recently described triphasic lipopolysaccharide (LPS) fevers have been repeatedly mistaken for biphasic fevers. Experiments were performed in loosely restrained male Wistar rats with a catheter implanted into the right jugular vein. Each animal was injected with Escherichia coli LPS, and its colonic (Tc) and tail skin temperatures were monitored. The results are presented as time graphs and phase-plane plots; in the latter case the rate of change of Tc is plotted against Tc. At an ambient temperature (Ta) of 30.0 degrees C, the response to the 10 microg/kg dose of LPS was triphasic, as is obvious from time graphs of Tc (3 peaks), time graphs of effector activity (3 waves of tail skin vasoconstriction), and phase-plane plots (3 complete loops). When the Ta was below neutral (22.0 degrees C) or the LPS dose was higher (100 or 1,000 microg/kg), the time graph of Tc did not allow for the reliable detection of all three febrile phases, but the phase-plane plot and time graph of effector activity clearly revealed the triphasic pattern. In a separate experiment, LPS (10 microg/kg) or saline was injected via one of two different procedures: in the first group the injection was performed through the jugular catheter, from outside the experimental chamber; in the second group the same nonstressing injection was combined with opening the chamber and pricking the animal in its lower abdomen with a needle. In the first group the febrile response was obviously triphasic, and none of the phases was due to the procedure of injection per se (injection of saline did not affect Tc). In the second group the fever similarly consisted of three Tc rises, but it might have been readily mistaken for biphasic because the first rise was indistinguishable from stress hyperthermia occurring in the saline-injected (and needle-pricked) controls. We conclude that several methodological factors (dose of LPS, procedure of its injection, and Ta) have contributed, although each in a different way, to the common misbelief that there are only two febrile phases.This study explains why the recently described triphasic lipopolysaccharide (LPS) fevers have been repeatedly mistaken for biphasic fevers. Experiments were performed in loosely restrained male Wistar rats with a catheter implanted into the right jugular vein. Each animal was injected with Escherichia coli LPS, and its colonic (Tc) and tail skin temperatures were monitored. The results are presented as time graphs and phase-plane plots; in the latter case the rate of change of Tc is plotted against Tc. At an ambient temperature (Ta) of 30.0°C, the response to the 10 μg/kg dose of LPS was triphasic, as is obvious from time graphs of Tc (3 peaks), time graphs of effector activity (3 waves of tail skin vasoconstriction), and phase-plane plots (3 complete loops). When the Ta was below neutral (22.0°C) or the LPS dose was higher (100 or 1,000 μg/kg), the time graph of Tc did not allow for the reliable detection of all three febrile phases, but the phase-plane plot and time graph of effector activity clearly revealed the triphasic pattern. In a separate experiment, LPS (10 μg/kg) or saline was injected via one of two different procedures: in the first group the injection was performed through the jugular catheter, from outside the experimental chamber; in the second group the same nonstressing injection was combined with opening the chamber and pricking the animal in its lower abdomen with a needle. In the first group the febrile response was obviously triphasic, and none of the phases was due to the procedure of injection per se (injection of saline did not affect Tc). In the second group the fever similarly consisted of three Tc rises, but it might have been readily mistaken for biphasic because the first rise was indistinguishable from stress hyperthermia occurring in the saline-injected (and needle-pricked) controls. We conclude that several methodological factors (dose of LPS, procedure of its injection, and Ta) have contributed, although each in a different way, to the common misbelief that there are only two febrile phases.

Multiple neural mechanisms of fever.

In rats, fevers induced by moderate-to-high doses of intravenous lipopolysaccharide consist of three phases (phases 1, 2 and 3) with body temperature peaks at approximately 1, 2, and 5 h postinjection, respectively. In this study, the effects of bilateral truncal subdiaphragmatic vagotomy and intraperitoneal capsaicin desensitization on febrile phases 1-3 were assessed in adult Wistar rats. Surgical vagotomy was performed approximately 30 d before the experiment; this procedure interrupts both afferent and efferent vagal fibers. Capsaicin was administered intraperitoneally in two consecutive injections (2 and 3 mg/kg, 3 h apart) 1 week prior to the experiment; this procedure desensitizes afferent fibers, primarily within the abdominal cavity, and does not lead to the known thermal effects of systemic capsaicin desensitization. At a neutral ambient temperature, the rats were given Escherichia coli lipopolysaccharide (10 microg/kg) through a preimplanted jugular catheter, and their colonic temperature wes measured by thermocouples for 7 h. The control rats exhibited the typical triphasic febrile responses. Confirming our earlier studies, subdiaphragmatic vagotomy did not affect phases 1 and 2; it did, however, result in a 2.5-fold reduction of phase 3. Capsaicin desensitization modified the febrile response differently: phases 2 and 3 were unaffected, but phase 1 disappeared. We suggest that neural afferent fibers (nonvagal but perhaps vagal as well) play an important role in the early febrile response (phase 1) by transducing peripheral pyrogenic signals to the brain. We also suggest that vagal efferent fibers are likely to participate in the later febrile response (phase 3) via an unknown mechanism.

“Biphasic” fevers often consist of more than two phases

This paper disproves the common belief that all doses of lipopolysaccharide (LPS) that are commonly referred to as biphasic fever inducing (>/=2 microg/kg) cause truly biphasic responses. A catheter was implanted into the right jugular vein of several strains of adult male rats, and the animals were habituated to the experimental conditions. At an ambient temperature of 30.0 degrees C, loosely restrained animals were injected with a 10 microg/kg dose of LPS (various preparations), and their colonic (Tc) and tail skin temperatures were monitored (from >/=1 h before to >/=7 h after the injection). The results are presented as time graphs and phase-plane plots; in the latter case the rate of change of Tc is plotted against Tc. In experiment 1 the intravenous injection of LPS (from Escherichia coli 0111:B4, phenol extract) into the rats (Bkl:Wistar) induced a triphasic febrile response, as is obvious from time graphs of Tc (3 peaks), time graphs of effector activity (3 waves of tail skin vasoconstriction), and phase-plane plots (3 complete loops); the injection of saline (control) induced no Tc changes. We analyzed whether the triphasic pattern was due to some peculiarities of the experimental design, i.e., the pyrogen preparation used (experiment 2) or the rat strain tested (experiment 3) or whether this pattern reflects a more general law. In experiment 2 we used the same (phenol) preparation of different LPS (from Shigella flexneri 1A and Salmonella typhosa) and a different preparation (TCA extract) of the same LPS (E. coli). Regardless of the LPS used, rats of the Bkl:Wistar strain responded to the 10 microg/kg dose with the triphasic fever. In experiment 3, rats of other strains [Bkl:Sprague-Dawley and Sim:(LE)fBR(Black-hooded)] were tested. Again, all animals responded to the 10 microg/kg dose of E. coli LPS (phenol extract) with the triphasic fever. Because all fevers caused by four different LPS preparations in three rat strains were triphasic, the triphasic pattern is likely to constitute an intrinsic characteristic of the febrile response.This paper disproves the common belief that all doses of lipopolysaccharide (LPS) that are commonly referred to as biphasic fever inducing (≥2 μg/kg) cause truly biphasic responses. A catheter was implanted into the right jugular vein of several strains of adult male rats, and the animals were habituated to the experimental conditions. At an ambient temperature of 30.0°C, loosely restrained animals were injected with a 10 μg/kg dose of LPS (various preparations), and their colonic (Tc) and tail skin temperatures were monitored (from ≥1 h before to ≥7 h after the injection). The results are presented as time graphs and phase-plane plots; in the latter case the rate of change of Tc is plotted against Tc. In experiment 1 the intravenous injection of LPS (from Escherichia coli 0111:B4, phenol extract) into the rats (Bkl:Wistar) induced a triphasic febrile response, as is obvious from time graphs of Tc (3 peaks), time graphs of effector activity (3 waves of tail skin vasoconstriction), and phase-plane plots (3 complete loops); the injection of saline (control) induced no Tc changes. We analyzed whether the triphasic pattern was due to some peculiarities of the experimental design, i.e., the pyrogen preparation used ( experiment 2) or the rat strain tested ( experiment 3) or whether this pattern reflects a more general law. In experiment 2 we used the same (phenol) preparation of different LPS (from Shigella flexneri 1A and Salmonella typhosa) and a different preparation (TCA extract) of the same LPS ( E. coli). Regardless of the LPS used, rats of the Bkl:Wistar strain responded to the 10 μg/kg dose with the triphasic fever. In experiment 3, rats of other strains [Bkl:Sprague-Dawley and Sim:(LE)fBR(Black-hooded)] were tested. Again, all animals responded to the 10 μg/kg dose of E. coli LPS (phenol extract) with the triphasic fever. Because all fevers caused by four different LPS preparations in three rat strains were triphasic, the triphasic pattern is likely to constitute an intrinsic characteristic of the febrile response.

Naturally occurring hypothermia is more advantageous than fever in severe forms of lipopolysaccharide- and Escherichia coli-induced systemic inflammation.

The natural switch from fever to hypothermia observed in the most severe cases of systemic inflammation is a phenomenon that continues to puzzle clinicians and scientists. The present study was the first to evaluate in direct experiments how the development of hypothermia vs. fever during severe forms of systemic inflammation impacts the pathophysiology of this malady and mortality rates in rats. Following administration of bacterial lipopolysaccharide (LPS; 5 or 18 mg/kg) or of a clinical Escherichia coli isolate (5 × 10(9) or 1 × 10(10) CFU/kg), hypothermia developed in rats exposed to a mildly cool environment, but not in rats exposed to a warm environment; only fever was revealed in the warm environment. Development of hypothermia instead of fever suppressed endotoxemia in E. coli-infected rats, but not in LPS-injected rats. The infiltration of the lungs by neutrophils was similarly suppressed in E. coli-infected rats of the hypothermic group. These potentially beneficial effects came with costs, as hypothermia increased bacterial burden in the liver. Furthermore, the hypotensive responses to LPS or E. coli were exaggerated in rats of the hypothermic group. This exaggeration, however, occurred independently of changes in inflammatory cytokines and prostaglandins. Despite possible costs, development of hypothermia lessened abdominal organ dysfunction and reduced overall mortality rates in both the E. coli and LPS models. By demonstrating that naturally occurring hypothermia is more advantageous than fever in severe forms of aseptic (LPS-induced) or septic (E. coli-induced) systemic inflammation, this study provides new grounds for the management of this deadly condition.

Improved Cellular Specificity of Plasmonic Nanobubbles versus Nanoparticles in Heterogeneous Cell Systems

The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level.

Lipopolysaccharide transport from the peritoneal cavity to the blood: is it controlled by the vagus nerve?

Vagotomy suppresses fever and hyperalgesia caused by intraperitoneal lipopolysaccharide (LPS) but has little effect on the febrile response to intravenous or intramuscular LPS. This suggests that some vagus-mediated mechanisms are recruited only when LPS is administered via the intraperitoneal route. We hypothesized that such mechanisms are associated with LPS transport from the peritoneal cavity to the circulation. Adult Wistar rats underwent total subdiaphragmatic, bilateral selective celiac, or sham vagotomy. On day 28-32 after surgery, they were injected IP with Escherichia coli LPS (5, 20, or 100 microg/kg) or saline and decapitated 90 min thereafter. Their plasma levels of LPS and their plasma interleukin-6, adrenocorticotropin, and corticosterone responses to LPS were measured. Success of intraperitoneal administration of LPS was verified by increased interleukin-1beta and interleukin-6 concentrations in the peritoneal lavage fluid. Effectiveness of vagotomies was confirmed by increased stomach mass (food retention) and pancreas mass (hypertrophy). In the shams, LPS caused a dose-dependent endotoxemia and increased plasma levels of interleukin-6, adrenocorticotropin, and corticosterone. Neither celiac nor total vagotomy affected any of these responses. LPS escapes from the peritoneal cavity by two primary routes, viz., the hematogenous (via the portal vein) and lymphogenous (via the lymphatic system). The design of the present study did not allow for evaluating the rapid, hematogenous transport. The results obtained suggest that the abdominal vagus does not control the slow. lymphogenous escape of LPS from the peritoneal cavity.

Does the formation of lipopolysaccharide tolerance require intact vagal innervation of the liver

The study was designed to test whether intact vagal innervation of the liver is required for the formation of tolerance to lipopolysaccharide (LPS). Wistar rats were subjected to either denervation of the liver (transection of the hepatic and both celiac branches of the abdominal vagus) or sham surgery. Two weeks later, each rat had an osmotic pump implanted subcutaneously. The pump was filled with either a suspension of Escherichia coli LPS (18 mg/ml) in saline or saline alone. Via a catheter, the pump delivered its content into the right jugular vein at a rate of approximately 0.72 microl/kg/h (approximately 13 microg/kg/h of LPS) over 28 d. On day 25 of the infusion, each animal had another catheter implanted into the left jugular vein. Three days later, each rat was injected with a lethal bolus dose of LPS (15 mg/kg) and had its colonic temperature recorded. The saline-infused sham-operated rats responded to the bolus injection of LPS with hypothermia followed by a fever (mean response magnitude 1.0+/-0.2 degrees C); 91% of the animals died within 24 h. The LPS-primed shams developed marked tolerance: When challenged with a lethal dose of LPS, they exhibited a significantly smaller thermal response (magnitude 0.5 +/- 0.2 degrees C) and none died. No group of the vagotomized animals, whether LPS- or saline-primed, became tolerant: Both groups exhibited similar hypothermic responses to the bolus LPS injection and a substantial mortality rate (40 and 100%, respectively). The study shows that prolonged infusion of low doses of LPS leads to the formation of tolerance and that vagal denervation of the liver by hepato-celiac vagotomy suppresses this process. The mechanisms of vagal control of the formation of LPS tolerance remain speculative.

Ongoing activity in trigeminal wide-dynamic range neurons is driven from the periphery.

Ongoing activity of spinal trigeminal neurons is observed under various conditions and suggested to be responsible for ongoing headache. It can be spontaneous, i.e. arising intrinsically from the neuron, or the product of descending influences from other central neurons, or maintained by ongoing afferent input. The aim of the present study was to examine if ongoing activity of neurons in different subnuclei of the spinal trigeminal nucleus is driven from peripheral afferent input. Experiments were performed in Wistar rats anesthetized with isoflurane or Nembutal/urethane. Ongoing activity of single wide-dynamic range (WDR) neurons was recorded with carbon fiber glass microelectrodes in two subnuclei of the spinal trigeminal nucleus: oral (Sp5O) and caudal (Sp5C). Peripheral receptive fields were evaluated using von Frey filaments. Sp5O neurons received peripheral input from facial areas innervated by the mandibular branch of the trigeminal nerve. Units in Sp5C had receptive fields in the surgically exposed dura mater and in facial areas innervated by the ophthalmic and maxillary branch of the trigeminal nerve. Saline or the local anesthetic lidocaine was locally applied onto the exposed dura mater or microinjected into V3 (for Sp5O units) or V1/V2 (for Sp5C units) divisions of the trigeminal ganglion via the infraorbital channel. Local application of lidocaine onto the exposed dura caused mechanical insensitivity of dural receptive fields but not significant decrease in ongoing activity. Microinjection of lidocaine but not saline into the trigeminal ganglion was followed by a substantial decrease in both the receptive field size and the activity of the recorded WDR units. Mechanical insensitivity of receptive fields after trigeminal ganglion blockade was accompanied by the disappearance of ongoing activity. We conclude that the ongoing activity of WDR neurons in the spinal trigeminal nucleus, which may be indicative for processes of sensitization, is driven remotely by ongoing afferent input.

Capsaicin-sensitive area in the ventral surface of the rat medulla

In acute experiments on nembutal-urethan-anesthetized rats, structures selectively sensitive to capsaicin were found near the ventral surface of the medulla at the exit of hypoglossal nerve roots. Microinjection of 5-50 nl 0.01% capsaicin to the rostral region of the capsaicin-sensitive area mostly activated respiration, arterial pressure and heart rate (HR) while that to the caudal region inhibited arterial pressure and HR. In chronic experiments on rats, injection of 25 nl 1% capsaicin to the caudal capsaicin-sensitive area led to a decrease in arterial pressure by 35-45% and in HR by 10-15% within a week after operation. Arterial pressure and HR virtually reached the control level and the rostral and caudal ventral medulla showed asymmetric distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive cells by the end of the 2nd week. It is suggested that nitric oxide may be involved in the mechanisms of neurochemical rearrangements in the brainstem after application of capsaicin to the caudal ventrolateral medulla (CVLM).