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Anterograde and retrograde tract tracing were combined with neurotransmitter and modulator immunolabeling to identify the chemical anatomy of vestibular nuclear neurons with direct projections to the solitary nucleus in rats. Direct, sparsely branched but highly varicose axonal projections from neurons in the caudal vestibular nuclei to the solitary nucleus were observed. The vestibular neurons giving rise to these projections were predominantly located in ipsilateral medial vestibular nucleus. The cell bodies were intensely glutamate immunofluorescent, and their axonal processes contained vesicular glutamate transporter 2, supporting the interpretation that the cells utilize glutamate for neurotransmission. The glutamate-immunofluorescent, retrogradely filled vestibular cells also contained the neuromodulator imidazoleacetic acid ribotide, which is an endogenous CNS ligand that participates in blood pressure regulation. The vestibulo-solitary neurons were encapsulated by axo-somatic GABAergic terminals, suggesting that they are under tight inhibitory control. The results establish a chemoanatomical basis for transient vestibular activation of the output pathways from the caudal and intermediate regions of the solitary nucleus. In this way, changes in static head position and movement of the head in space may directly influence heart rate, blood pressure, respiration, as well as gastrointestinal motility. This would provide one anatomical explanation for the synchronous heart rate and blood pressure responses observed after peripheral vestibular activation, as well as disorders ranging from neurogenic orthostatic hypotension, postural orthostatic tachycardia syndrome, and vasovagal syncope to the nausea and vomiting associated with motion sickness. NEW & NOTEWORTHY Vestibular neurons with direct projections to the solitary nucleus utilize glutamate for neurotransmission, modulated by imidazoleacetic acid ribotide. This is the first direct demonstration of the chemical neuroanatomy of the vestibulo-solitary pathway.

The vestibulosympathetic reflex (VSR) increases blood pressure (BP) upon arising to maintain blood flow to the brain. The optimal directions of VSR activation and whether changes in heart rate (HR) are associated with changes in BP are still not clear. We used manually activated pulses and oscillatory linear accelerations of 0.2–2.5 g along the naso-occipital, interaural, and dorsoventral axes in isoflurane-anesthetized, male Long-Evans rats. BP and HR were recorded with an intra-aortic sensor and acceleration with a three-dimensional accelerometer. Linear regressions of BP changes in accelerations along the upward, downward, and forward axes had slopes of ≈3–6 mmHg · g−1( P < 0.05). Lateral and backward accelerations did not produce consistent changes in BP. Thus upward, downward, and forward translations were the directions that significantly altered BP. HR was unaffected by these translations. The VSR sensitivity to oscillatory forward-backward translations was ≈6–10 mmHg · g−1at frequencies of ≈0.1 Hz (0.2 g), decreasing to zero at frequencies above 2 Hz (1.8 g). Upward, 70° tilts of an alert rat increased BP by 9 mmHg · g−1without changes in HR, indicating that anesthesia had not reduced the VSR sensitivity. The similarity in BP induced in alert and anesthetized rats indicates that the VSR is relatively insensitive to levels of alertness and that the VSR is likely to cause changes in BP through modification of peripheral vascular resistance. Thus the VSR, which is directed toward the cardiovascular system, is in contrast to the responses in the alert state that can produce sweating, alterations in BP and HR, and motion sickness.

Imidazole-4-acetic acid-ribotide (IAARP) is a putative neurotransmitter/modulator and an endogenous regulator of sympathetic drive, notably systemic blood pressure, through binding to imidazoline receptors. IAARP is present in neurons and processes throughout the CNS, but is particularly prevalent in regions that are involved in blood pressure control. The goal of this study was to determine whether IAARP is present in neurons in the caudal vestibular nuclei that participate in the vestibulo-sympathetic reflex (VSR) pathway. This pathway is important in modulating blood pressure upon changes in head position with regard to gravity, as occurs when humans rise from a supine position and when quadrupeds climb or rear. Sinusoidal galvanic vestibular stimulation was used to activate the VSR and cfos gene expression in VSR pathway neurons of rats. These subjects had previously received a unilateral FluoroGold tracer injection in the rostral or caudal ventrolateral medullary region. The tracer was transported retrogradely and filled vestibular neuronal somata with direct projections to the injected region. Brainstem sections through the caudal vestibular nuclei were immunostained to visualize FluoroGold, cFos protein, IAARP and glutamate immunofluorescence. The results demonstrate that IAARP is present in vestibular neurons of the VSR pathway, where it often co-localizes with intense glutamate immunofluorescence. The co-localization of IAARP and intense glutamate immunofluorescence in VSR neurons may represent an efficient chemoanatomical configuration, allowing the vestibular system to rapidly up- and down-modulate the activity of presympathetic neurons in the ventrolateral medulla, thereby altering blood pressure.

Vasovagal syncope is a significant medical problem without effective therapy, postulated to be related to a collapse of baroreflex function. While some studies have shown that repeated static tilts can block vasovagal syncope, this was not found in other studies. Using anesthetized, male Long–Evans rats that were highly susceptible to generation of vasovagal responses, we found that repeated activation of the vestibulosympathetic reflex (VSR) with ±2 and ±3 mA, 0.025 Hz sinusoidal galvanic vestibular stimulation (sGVS) caused incremental changes in blood pressure (BP) and heart rate (HR) that blocked further generation of vasovagal responses. Initially, BP and HR fell ≈20–50 mmHg and ≈20–50 beats/min (bpm) into a vasovagal response when stimulated with Sgv\S in susceptible rats. As the rats were continually stimulated, HR initially rose to counteract the fall in BP; then the increase in HR became more substantial and long lasting, effectively opposing the fall in BP. Finally, the vestibular stimuli simply caused an increase in BP, the normal sequence following activation of the VSR. Concurrently, habituation caused disappearance of the low-frequency (0.025 and 0.05 Hz) oscillations in BP and HR that must be present when vasovagal responses are induced. Habituation also produced significant increases in baroreflex sensitivity (p

Changes in head position and posture are detected by the vestibular system and are normally followed by rapid modifications in blood pressure. These compensatory adjustments, which allow humans to stand up without fainting, are mediated by integration of vestibular system pathways with blood pressure control centers in the ventrolateral medulla. Orthostatic hypotension can reflect altered activity of this neural circuitry. Vestibular sensory input to the vestibulo-sympathetic pathway terminates on cells in the vestibular nuclear complex, which in turn project to brainstem sites involved in the regulation of cardiovascular activity, including the rostral and caudal ventrolateral medullary regions (RVLM and CVLM, respectively). In the present study, sinusoidal galvanic vestibular stimulation was used to activate this pathway, and activated neurons were identified through detection of c-Fos protein. The retrograde tracer FluoroGold was injected into the RVLM or CVLM of these animals, and immunofluorescence studies of vestibular neurons were conducted to visualize c-Fos protein and FluoroGold concomitantly. We observed activated projection neurons of the vestibulo-sympathetic reflex pathway in the caudal half of the spinal, medial and parvocellular medial vestibular nuclei. Approximately two-thirds of the cells were ipsilateral to FluoroGold injection sites in both RVLM and CVLM and the remainders were contralateral. As a group, cells projecting to RVLM were located slightly rostral to those with terminals in CVLM. Individual activated projection neurons were multipolar, globular or fusiform in shape. This study provides the first direct demonstration of the central vestibular neurons that mediate the vestibulo-sympathetic reflex.

Traumatic brain injury (TBI) is a major public health issue in the USA, with over 1.7 million cases per year. At least 5.3 million Americans currently live with ongoing disability due to TBI (Walker and Tesco 2013). The long-term consequences of TBI can be complex and progressive: 10–15% of individuals diagnosed with mild TBI continue to suffer from persistent symptoms, while as many as 50% of patients with moderate TBI experience long-term dysfunction (Bales et al. 2009; Walker and Tesco 2013). In addition to the post-injury development of cognitive deficits in attention, memory, and executive function (Bales et al. 2009), a chronic inflammatory state can persist in the brain for months, and even years, following TBI (Opii et al. 2007; Piao et al. 2013). Despite the prevalence of these chronic dysfunctions following TBI, elucidating the mechanisms that underlie these symptoms has proven challenging. It is understood that the post-injury processes of TBI involve a primary and a secondary phase of injury. Primary injury occurs during the initial insult, resulting from the displacement of the physical structures within the brain. Secondary injury occurs over time and involves an interdependent series of cellular dysfunctions that persist long after injury (Giza and Hovda 2001; Nilsson et al. 1996; Park et al. 2008; Walker and Tesco 2013; Werner and Engelhard 2007). Currently, it is not well understood how the acute mechanical injury of TBI, and subsequent secondary injury processes, can result in serious long-term dysfunctions that can persist for years.

The vestibular system senses the movement and position of the head in space and uses this information to stabilize vision, control posture, perceive head orientation and self-motion in three-dimensional space, and modulate autonomic and limbic activity in response to locomotion and changes in posture. Most vestibular signals are not consciously perceived and are usually appreciated through effector pathways classically described as the vestibulo-ocular, vestibulo-spinal, vestibulo-collic and vestibulo-autonomic reflexes. The present study reviews some of the recent data concerning the connectivity and chemical anatomy of vestibular projections to autonomic sites that are important in the sympathetic control of blood pressure.

Blood pressure (BP) and heart rate (HR) were studied in isoflurane-anesthetized Long-Evans rats during sinusoidal galvanic vestibular stimulation (sGVS) and sinusoidal oscillation in pitch to characterize vestibular influences on autonomic control of BP and HR. sGVS was delivered binaurally via Ag/AgCl needle electrodes inserted over the mastoids at stimulus frequencies 0.008-0.4 Hz. Two processes affecting BP and HR were induced by sGVS: 1) a transient drop in BP (≈15-20 mmHg) and HR (≈3 beat*s(-1)), followed by a slow recovery over 1-6 min; and 2) inhibitory modulations in BP (≈4.5 mmHg/g) and HR (≈0.15 beats*s(-1)/g) twice in each stimulus cycle. The BP and HR modulations were approximately in-phase with each other and were best evoked by low stimulus frequencies. A wavelet analysis indicated significant energies in BP and HR at scales related to twice and four times the stimulus frequency bands. BP and HR were also modulated by oscillation in pitch at frequencies 0.025-0.5 Hz. Sensitivities at 0.025 Hz were ≈4.5 mmHg/g (BP) and ≈0.17 beat*s(-1)/g (HR) for pitches of 20-90°. The tilt-induced BP and HR modulations were out-of-phase, but the frequencies at which responses were elicited by tilt and sGVS were the same. The results show that the sGVS-induced responses, which likely originate in the otolith organs, can exert a powerful inhibitory effect on both BP and HR at low frequencies. These responses have a striking resemblance to human vasovagal responses. Thus, sGVS-activated rats can potentially serve as a useful experimental model of the vasovagal response in humans.

Imidazole-4-acetic acid-ribotide (IAA-RP), an endogenous agonist at imidazoline receptors (I-Rs), is a putative neurotransmitter/regulator in mammalian brain. We studied the effects of IAA-RP on excitatory transmission by performing extracellular and whole cell recordings at Schaffer collateral-CA1 synapses in rat hippocampal slices. Bath-applied IAA-RP induced a concentration-dependent depression of synaptic transmission that, after washout, returned to baseline within 20 min. Maximal decrease occurred with 10 μM IAA-RP, which reduced the slope of field extracellular postsynaptic potentials (fEPSPs) to 51.2 ± 5.7% of baseline at 20 min of exposure. Imidazole-4-acetic acid-riboside (IAA-R; 10 μM), the endogenous dephosphorylated metabolite of IAA-RP, also produced inhibition of fEPSPs. This effect was smaller than that produced by IAA-RP (to 65.9 ± 3.8% of baseline) and occurred after a further 5- to 8-min delay. The frequency, but not the amplitude, of miniature excitatory postsynaptic currents was decreased, and paired-pulse facilitation (PPF) was increased after application of IAA-RP, suggesting a principally presynaptic site of action. Since IAA-RP also has low affinity for α2-adrenergic receptors (α2-ARs), we tested synaptic depression induced by IAA-RP in the presence of α2-ARs, I1-R, or I3-R antagonists. The α2-AR antagonist rauwolscine (100 nM), which blocked the actions of the α2-AR agonist clonidine, did not affect either the IAA-RP-induced synaptic depression or the increase in PPF. In contrast, efaroxan (50 μM), a mixed I1-R and α2-AR antagonist, abolished the synaptic depression induced by IAA-RP and abolished the related increase in PPF. KU-14R, an I3-R antagonist, partially attenuated responses to IAA-RP. Taken together, these data support a role for IAA-RP in modulating synaptic transmission in the hippocampus through activation of I-Rs.

While the basic pathways mediating vestibulo-ocular, -spinal, and -collic reflexes have been described in some detail, little is known about vestibular projections to central autonomic sites. Previous studies have primarily focused on projections from the caudal vestibular region to solitary, vagal and parabrachial nuclei, but have noted a sparse innervation of the ventrolateral medulla. Since a direct pathway from the vestibular nuclei to the rostral ventrolateral medulla would provide a morphological substrate for rapid modifications in blood pressure, heart rate and respiration with changes in posture and locomotion, the present study examined anatomical evidence for this pathway using anterograde and retrograde tract tracing and immunofluorescence detection in brainstem sections of the rat medulla. The results provide anatomical evidence for direct pathways from the caudal vestibular nuclear complex to the rostral and caudal ventrolateral medullary regions. The projections are conveyed by fine and highly varicose axons that ramify bilaterally, with greater terminal densities present ipsilateral to the injection site and more rostrally in the ventrolateral medulla. In the rostral ventrolateral medulla, these processes are highly branched and extremely varicose, primarily directed toward the somata and proximal dendrites of non-catecholaminergic neurons, with minor projections to the distal dendrites of catecholaminergic cells. In the caudal ventrolateral medulla, the axons of vestibular nucleus neurons are more modestly branched, with fewer varicosities, and their endings are contiguous with both the perikarya and dendrites of catecholamine-containing neurons. These data suggest that vestibular neurons preferentially target the rostral ventrolateral medulla, and can thereby provide a morphological basis for a short latency vestibulo-sympathetic pathway.

The development and cellular distribution of the inhibitory neurotransmitter glycine in the spinal cord of the sea lamprey were studied by immunocytochemistry and double immunofluorescence and compared with the distribution of γ-aminobutyric acid (GABA). Results in lamprey embryos and prolarvae reveal that the appearance of glycine-immunoreactive (-ir) spinal neurons precedes that of GABA-ir neurons. Throughout development, glycine-ir cells in the lateral and dorsomedial gray matter of the spinal cord are more numerous than the GABA-ir cells. Only a subset of these neurons shows colocalization of GABA and glycine, suggesting that they are primarily disparate neuronal populations. In contrast, most cerebrospinal fluid (CSF)-contacting neurons of the central canal walls are strongly GABA-ir, and only a portion of them are faintly glycine-ir. Some edge cells (lamprey intraspinal mechanoreceptors) were glycine-ir in larvae and adults. The glycine-ir and GABA-ir neuronal populations observed in the adult spinal cord were similar to those found in larvae. Comparison of glycine-ir and GABA-ir fibers coursing longitudinally in the spinal cord of adult lamprey revealed large differences in diameter between these two types of fiber. Commissural glycine-ir fibers appear in prolarvae and become numerous at larval stages, whereas crossed GABA-ir are scarce. Taken together, results in this primitive vertebrate indicate that the spinal glycinergic cells do not arise by biochemical shift of preexisting GABAergic cells but instead suggest that glycine is present in the earliest circuitry of the developing lamprey spinal cord, where it might act transiently as an excitatory transmitter. J. Comp. Neurol. 508:112–130, 2008. © 2008 Wiley-Liss, Inc.

Nitric oxide is an unstable free radical that serves as a novel messenger molecule in the central nervous system (CNS). In order to understand the interplay between classic and novel chemical communication systems in vestibular pathways, the staining obtained using a monoclonal antibody directed against L-citrulline was compared with the labeling observed using more traditional markers for the presence of nitric oxide. Brainstem tissue from adult rats was processed for immunocytochemistry employing a monoclonal antibody directed against L-citrulline, a polyclonal antiserum against neuronal nitric oxide synthase, and/or NADPH-diaphorase histochemistry. Our findings demonstrate that L-citrulline can be fixed in situ by vascular perfusion, and can be visualized in fixed CNS tissue sections by immunocytochemistry. Further, the same vestibular regions and cell types are labeled by NADPH-diaphorase histochemistry, by the neuronal nitric oxide synthase antiserum, and by our anti-L-citrulline antibody. Clusters of L-citrulline-immunoreactive neurons are present in subregions of the vestibular nuclei, including the caudal portion of the inferior vestibular nucleus, the magnocellular portion of the medial vestibular nucleus, and the large cells in the ventral tier of the lateral vestibular nucleus. NADPH-diaphorase histochemical staining of these neurons clearly demonstrated their multipolar, fusiform and globular somata and long varicose dendritic processes. These results provide support for the suggestion that nitric oxide serves key roles in both vestibulo-autonomic and vestibulo-spinal pathways.

The vestibular semicircular canals respond to angular acceleration that is integrated to angular velocity by the biofluid mechanics of the canals and is the primary origin of afferent responses encoding velocity. Surprisingly, some afferents actually report angular acceleration. Our data indicate that hair-cell/afferent synapses introduce a mathematical derivative in these afferents that partially cancels the biomechanical integration and results in discharge rates encoding angular acceleration. We examined the role of convergent synaptic inputs from hair cells to this mathematical differentiation. A significant reduction in the order of the differentiation was observed for low-frequency stimuli after γ-aminobutyric acid type B receptor antagonist administration. Results demonstrate that γ-aminobutyric acid participates in shaping the temporal dynamics of afferent responses.

The present study was conducted to visualize the ultrastructural features of vestibular efferent boutons in the oyster toadfish, Opsanus tau. The crista ampullaris of the horizontal semicircular canal was processed for and examined by routine transmission electron microscopy. The results demonstrate that such boutons vary in size and shape, and contain a heterogeneous population of lucent vesicles with scattered dense core vesicles. Efferent contacts with hair cells are characterized by local vesicle accumulations in the presynaptic terminal and a subsynaptic cistern in the postsynaptic region of the hair cell. Serial efferent to hair cell to afferent synaptic arrangements are common, particularly in the central portion of the crista. However, direct contacts between efferent terminals and afferent neurites were not observed in our specimens. The existence of serial synaptic contacts, often with a row of vesicles in the efferent boutons lining the efferent-afferent membrane apposition, suggests that the efferent influence on the crista may involve both synaptic and nonsynaptic, secretory mechanisms. Further, it is suggested that differences in more subtle aspects of synaptic architecture and/or transmitter and receptor localization and interaction may render the efferent innervation of the peripheral crista less effective in influencing sensory processing.

Although γ-aminobutyric acid (GABA) and glutamate are known to be present in the vestibular sensory epithelia of a variety of species, the functional relationship between these two transmitters is not clear. The present study addresses the three-dimensional spatial distribution of GABA and glutamate immunoreactivity in the vestibular labyrinth of the oyster toadfish by using whole end organs labeled by immunofluorescence with monoclonal anti-GABA and/or antiglutamate antibodies and visualized as whole mounts by multiphoton confocal microscopy. We find glutamate-immunoreactive hair cells present throughout the sensory epithelium. In contrast, prominent GABA immunoreactivity is restricted to a small population of hair cells located in the central region of the crista. Double immunofluorescence reveals two distinct staining patterns in GABA-labeled hair cells. Most (∼80%) GABA-labeled cells show trace levels of glutamate, appropriate for the metabolic/synthetic role of cytoplasmic glutamate. The remainder of the GABA-stained cells contain substantial levels of both GABA and glutamate, suggesting transmitter colocalization. In the toadfish utricle, glutamatergic hair cells are present throughout the macula. GABA-immunoreactive hair cells follow the arc of the striola, and most GABA-labeled receptor cells coexpress glutamate. The localization of GABA was explored in other species as well. In the pigeon, GABAergic hair cells are present throughout the crista ampullaris. Our findings demonstrate that multiple, neurochemically distinct types of hair cells are present in vestibular sensory epithelia. These observations, together with the excitatory activity generally associated with 8th nerve afferent fibers, strongly suggest that GABA serves an important, specific, and complex role in determining primary afferent response dynamics. J. Comp. Neurol. 471:1–10, 2004. © 2004 Wiley-Liss, Inc.

We have previously reported that CTL were demonstrable early after inoculation of CMS5 fibrosarcoma cells, but that they disappeared within 3 wk. These mice were unable to reject a challenge with CMS5 tumor cells. Other studies demonstrated cell surface phenotype and signaling abnormalities of cells within the spleen. Since we assumed that such an environment would make it more difficult to elicit antitumor immune responses via immunotherapy, we asked whether resection of the tumor could reverse these abnormalities. Although early after tumor cell inoculation tumor resection leads to the development of immunity, the effect at late time points has not been studied critically. To test this, mice were inoculated s.c. with CMS5 cells and after 28 days the tumors were resected. We observed a gradual normalization of the cellular phenotype of the spleen. In particular, there was a decrease in the number of Mac1+/Gr1high cells and an increase in the number of CD3+ cells in the spleen within 24–48 h of tumor resection. By day 10, these values were normal. Levels of p56lck increased as well. The functional implications of these changes were illustrated by the reduced growth rate or the complete rejection of a challenge of tumor cells in the resected mice. Both CD4+ and CD8+ cells were involved in the restoration of tumor immunity. Our results suggested that tumor resection not only led to the reversal of immune suppression, but also unmasked a population of primed T cells able to mediate protective immunity.

The purpose of the present study was to visualize the synaptic interactions of GABAergic neurons involved in the mediation of velocity storage. In the previous report, ultrastructural studies of degenerating neurons were conducted following midline section of rostral medullary commissural fibers with subsequent behavioral testing. The midline lesion caused functionally discrete damage to the velocity storage component, but not to the direct pathway, of the angular vestibulo-ocular reflex, and the degenerating neurons were interpreted as potential participants in the velocity storage network. We concluded that at least some of the commissural axons mediating velocity storage originate from clusters of neurons in the lateral crescents of the rostral medial vestibular nucleus. In the present report, immunocytochemical evidence is presented that many vestibular commissural neurons, putatively involved in mediating velocity storage, are GABAergic. These cells have large nuclei, small round or narrow tubular mitochondria, occasional cisterns and vacuoles, but few other organelles. Their axons are thinly-myelinated, and terminate in boutons containing mitochondria of similar ultrastructural appearance and a moderate density of round/pleomorphic synaptic vesicles. Such terminals often form axoaxonic synapses, and less frequently axodendritic contacts, with non-GABAergic elements. On the basis of the present results, we conclude that a portion of the commissural neurons of the velocity storage pathway is GABAergic. The observation of GABAergic axoaxonic synapses in this pathway is interpreted as a structural basis for presynaptic inhibition of medial vestibular nucleus circuits by velocity storage-related commissural neurons. Conversely, substantial ultrastructural evidence for postsynaptic inhibition of non-GABAergic commissural cells argues for a dual role for GABAergic terminals mediating velocity storage: presynaptic inhibition of non-GABAergic vestibular cells by GABAergic velocity storage commissural axons, and postsynaptic inhibition of non-GABAergic velocity storage cells by GABAergic axons. Both pre- and postsynaptic inhibitory arrangements could provide the morphologic basis for disinhibitory activation of the velocity storage network within local neuronal circuits.

Background. The pathogenesis of chronic rejection likely involves an interplay between immunogenic and nonimmunogenic factors. The objective of this study was to determine the influence of cold ischemic preservation injury on the rate of progression to chronic rejection in the Lewis to F344 cardiac allograft model. Methods. To induce an ischemic injury, donor hearts were stored for 3 hr at 4°C in University of Wisconsin solution before transplantation. Allografts were excised at 1, 7, and 90 days after transplantation or at rejection. Vasculopathy was graded for degree of inti-mal thickening based on the involvement of vascular perimeter and luminal compromise. Results. The degree of vessel injury in ischemic injured allografts at 90 days was significantly greater than in nonischemic injured allografts (2.8±0.4 vs. 1.6±0.5, P

Vasovagal responses (VVRs) are characterized by transient drops in blood pressure (BP) and heart rate (HR) and increased amplitude of low-frequency oscillations in the Mayer wave frequency range. Typical VVRs were induced in anesthetized, male, Long-Evans rats by sinusoidal galvanic vestibular stimulation (sGVS). VVRs were also produced by single sinusoids that transiently increased BP and HR, by 70–90° nose-up tilts, and by 60° tilts of the gravitoinertial acceleration vector using translation while rotating (TWR). The average power of the BP signal in the Mayer wave range increased substantially when tilts were >70° (0.91 g), i.e., when linear accelerations in the x–z plane were ≥0.9–1.0 g. The standard deviations of the wavelet-filtered BP signals during tilt and TWR overlaid when they were normalized to 1 g. Thus, the amplitudes of the Mayer waves coded the magnitude of the linear acceleration ≥1 g acting on the head and body, and the average power in this frequency range was associated with the generation of VVRs. These data show that VVRs are a natural outcome of stimulation of the vestibulosympathetic reflex and are not a disease. The results also demonstrate the usefulness of the rat as a small animal model for studying human VVRs.—Cohen, B., Martinelli, G. P., Raphan, T., Schaffner, A., Xiang, Y., Holstein, G. R., Yakushin, S. B. The vasovagal response of the rat: its relation to the vestibulosympathetic reflex and to Mayer waves.

The induction of donor-specific unresponsiveness in allograft recipients would lessen the need for chronic immunosuppression and its concomitant morbidities. In view of recognized interactions between the immune and neuroendocrine systems, we hypothesized that manipulating prolactin (PRL) levels might enhance the immunosuppressive effects of donor-specific blood transfusions. Bromocriptine (BR) and domperidone (DOM), administered via osmotic pumps, were used to inhibit or increase pituitary PRL secretion, respectively, in male LEW rats treated with donor-specific transfusions (DST, Day -1), cyclosporine (CsA, 5 mg/kg, Days -1 to +1), and receiving ACI heart allografts. Neither compound had direct effects on lymphoid cells in vitro. BR had no effects on graft survival in rats treated with either BT or CsA (BR-DST, 7.0 +/- 0.7; BR-CsA, 9.2+/-3.1; CsA, 11.3 +/- 3.9 days). DOM-DST-CsA also did not affect graft survival (8.7 +/- 3.1 days). BR and CsA, similarly, had no effects in rats receiving a nonspecific transfusion (8.8 +/- 1.1 days). In contrast, BR administration in rats treated with DST and CsA unequivocally prolonged graft survival (17.0 +/- 1.4 days; P0.01 vs all controls), suggesting that hypoprolactinemia increased the tolerogenic effects of DST. Spleen and lymph node cells harvested from BR-DST-CsA rats on Postoperative Day 8 showed impaired responses to mitogenic or allogeneic challenges. Cytotoxic antibody levels at Day 5 were low in all groups receiving CsA. Possible mechanisms are discussed.

This study evaluates the ability of the immunosuppressive drugs dexamethasone, cyclosporine, FK506 and rapamycin, alone and in combination to suppress interleukin-1 beta (IL-1 beta) secretion in vitro by THP-1 cells when stimulated by lipopolysaccharide. All four drugs, when added to cell culture medium at therapeutic concentrations, significantly decrease secretion of the monokine to well below control levels. However, only dexamethasone completely suppresses IL-1 beta secretion in a dose-dependent fashion. Cyclosporine, FK506 and rapamycin only partially suppress secretion of IL-1 beta at concentrations within their therapeutic ranges and increasing concentrations of the drugs do not result in further suppression of secretion. Likewise, the combination of any two of these three drugs does not provide any additional suppressive effect. Dexamethasone, however, when added in increasing concentrations in combination with any of the other drugs, results in further suppression of IL-1 secretion in a dose-dependent fashion. These data suggest that cyclosporine, FK506 and rapamycin all share a common effect on the production of IL-1 beta, different from that of dexamethasone.

The vestibular system sends projections to brainstem autonomic nuclei that modulate heart rate and blood pressure in response to changes in head and body position with regard to gravity. Consistent with this, binaural sinusoidal galvanic vestibular stimulation (sGVS) in humans causes vasoconstriction in the legs, while low frequency (0.02-0.04 Hz) sGVS causes a rapid drop in heart rate and blood pressure in anesthetized rats. We have hypothesized that these responses occur through activation of vestibulo-sympathetic pathways. In the present study, c-Fos protein expression was examined in neurons of the vestibular nuclei and rostral ventrolateral medullary region (RVLM) that were activated by low frequency sGVS. We found c-Fos-labeled neurons in the spinal, medial and superior vestibular nuclei (SpVN, MVN and SVN, respectively) and the parasolitary nucleus. The highest density of c-Fos-positive vestibular nuclear neurons was observed in MVN, where immunolabeled cells were present throughout the rostro-caudal extent of the nucleus. C-Fos expression was concentrated in the parvocellular region and largely absent from magnocellular MVN. C-Fos-labeled cells were scattered throughout caudal SpVN, and the immunostained neurons in SVN were restricted to a discrete wedge-shaped area immediately lateral to the IVth ventricle. Immunofluorescence localization of c-Fos and glutamate revealed that approximately one third of the c-Fos-labeled vestibular neurons showed intense glutamate-like immunofluorescence, far in excess of the stain reflecting the metabolic pool of cytoplasmic glutamate. In the RVLM, which receives a direct projection from the vestibular nuclei and sends efferents to preganglionic sympathetic neurons in the spinal cord, we observed an approximately 3-fold increase in c-Fos labeling in the sGVS-activated rats. We conclude that localization of c-Fos protein following sGVS is a reliable marker for sGVS-activated neurons of the vestibulo-sympathetic pathway.

We followed the development of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) system during locust embryogenesis in whole mount nervous systems and brain sections by using various cytochemical techniques. We visualized NO-sensitive neurons by cGMP immunofluorescence after incubation with an NO donor in the presence of the soluble guanylyl cyclase (sGC) activator YC-1 and the phosphodiesterase-inhibitor isobutyl-methyl-xanthine (IBMX). Central nervous system (CNS) cells respond to NO as early as 38% embryogenesis. By using the NADPH-diaphorase technique, we identified somata and neurites of possible NO-synthesizing cells in the CNS. The first NADPH-diaphorase-positive cell bodies appear around 40% embryogenesis in the brain and at 47% in the ventral nerve cord. The number of positive cells reaches the full complement of adult cells at 80%. In the brain, some structures, e.g., the mushroom bodies acquire NADPH-diaphorase staining only postembryonically. Immunolocalization of L-citrulline confirmed the presence of NOS in NADPH-diaphorase-stained neurons and, in addition, indicated enzymatic activity in vivo. In whole mount ventral nerve cords, citrulline immunolabeling was present in varying subsets of NADPH-diaphorase-positive cells, but staining was very variable and often weak. However, in a regeneration paradigm in which one of the two connectives between ganglia had been crushed, strong, reliable staining was observed as early as 60% embryogenesis. Thus, citrulline immunolabeling appears to reflect specific activity of NOS. However, in younger embryos, NOS may not always be constitutively active or may be so at a very low level, below the citrulline antibody detection threshold. For the CNS, histochemical markers for NOS do not provide conclusive evidence for a developmental role of this enzyme.

Fifty-two patients with ulcerative colitis and colorectal cancer undergoing colectomy at the Mount Sinai Hospital between 1973 and 1988 were studied retrospectively to determine the correlation of age, sex, duration of colitis, tumor location, number of cancers, tumor differentiation, colloid content, presence of signet ring cells, Dukes' classification, and DNA ploidy with survival. The mean age was 45 years, with a mean duration of colitis of 21 years. Five patients (10%) had Dukes' A lesions, 17 (33%) had Dukes' B lesions, 17 (33%) had Dukes' C lesions, and 13 (25%) had distant metastases. Thirty patients (58%) had well- or moderately differentiated tumors, whereas tumors were poorly differentiated in 22 (42%). Twenty-eight patients (54%) had colloid tumors, and, in 14 (27%), signet ring cells were present. Thirty-one patients (60%) had nondiploid tumors. Actuarial analysis revealed that the 5-year survival rate was significantly worse for patients with nondiploid tumors (76% versus 32%). When stratified by stage, only patients with Dukes' C lesions showed a significant difference in survival for diploid versus nondiploid tumors. Multivariate analysis showed that the Dukes' classification was the best prognostic indicator, followed by tumor differentiation and DNA ploidy. Tumor location, colloid content, number of cancers, duration of disease, age, and sex did not correlate with the prognosis.

L-Baclofen-sensitive GABAB binding sites in the medial vestibular nucleus (MVN) were identified immunocytochemically and visualized ultrastructurally in L-baclofen-preinjected rats and monkeys, using a mouse monoclonal antibody with specificity for the p-chlorophenyl moiety of baclofen. Saline-preinjected animals showed no immunostain. In drug-injected animals, there was evidence for both pre- and postsynaptic GABAergic inhibition in MVN mediated by GABAB receptors. These neural elements could be utilized in control of velocity storage in the vestibulo-ocular reflex.

Monoclonal antibodies were raised against the L-enantiomer of baclofen conjugated by glutaraldehyde to keyhole limpet hemocyanin. Hybridoma clones were selected for their stability and their production of high titers of antibodies directed against the p-chlorophenyl moiety of the L-baclofen molecule. The chosen antibody showed no cross-reactivity with conjugates of GABA and other neurotransmitters to human or bovine serum albumin. Specificity was further confirmed by the ability of L-baclofen-HCl to inhibit the binding of the antibody to L-baclofen-bovine serum albumin conjugate. Immunocytochemical studies were conducted on brain tissue from rats and monkeys injected with baclofen to localize baclofen-sensitive GABAB receptor sites. In these animals, the molecular layer of cerebellar cortex was clearly immunostained and the granular layer showed only some pale immunoreactivity. Ultrastructural observations were conducted in cerebellar cortex, as well as in the substantia nigra and the vestibular nuclei. Discrete labeling of neuronal profiles was observed in these structures, and both immunoperoxidase and colloidal gold methods were employed successfully. Material from saline-injected control animals showed no immunoreactivity at both light and electron microscopic levels. We conclude that the anti-L-baclofen antibody preferentially recognizes the p-chlorophenyl moiety of the baclofen molecule. Antibodies of such specificity are useful tools for the ultrastructural localization of baclofen-sensitive GABAB receptor sites. In general, antibodies directed against accessible moieties of specific neuroactive substances may serve as valuable markers for their sites of action.

A substantial body of research has led to the recognition that the vestibular system participates in blood pressure modulation during active movements and changes in posture, and that this modulation is effected at least partly by the caudal vestibular nuclei. The I-4 isomer of imidazoleacetic acid-ribotide (IAA-RP) is a putative neurotransmitter/modulator that is thought to be an endogenous regulator of general sympathetic drive, particularly systemic blood pressure. The present study employed immunofluorescence and light and electron microscopic immunocytochemistry to visualize IAA-RP in the vestibular nuclei of adult male rats. The results demonstrate IAA-RP immunolabeling of subpopulations of vestibular neurons in the descending nucleus and the caudal half of the medial nucleus, with scattered immunostained vestibular neurons also present more rostrally. On the basis of double immunofluorescence staining for IAA-RP and calbindin, many of these ribotide-immunoreactive neurons appear to be innervated by cerebellar Purkinje cell afferents. Ultrastructural observations in the caudal vestibular nuclei confirm the IAA-RP immunolocalization in cell bodies and dendritic processes, and in some myelinated axons and presynaptic boutons. The regional distribution of IAA-RP immunoreactivity corresponds to the location of vestibular neurons involved in autonomic functions. The presence of IAA-RP in those neurons suggests that they participate specifically in vestibulo-autonomic regulation of blood pressure. The localization of immunostain in processes and terminals suggests that vestibulo-autonomic activity is subject to local feedback control. Overall, the observations offer a chemoanatomic basis for understanding the vestibular side effects commonly experienced by patients treated with clonidine and other imidazoline-related drugs.

Imidazoleacetic acid-ribotide (IAA-RP) is a putative neurotransmitter/modulator recently discovered in mammalian brain. The present study examines the distribution of IAA-RP in the rat CNS using a highly specific antiserum raised in rabbit against IAA-RP with immunostaining of aldehyde-fixed rat CNS. IAA-RP-immunoreactive neurons were present throughout the neuraxis; neuroglia were not labeled. In each region, only a subset of the neuronal pool was immunostained. In the forebrain, ribotide-immunolabeled neurons were common in neocortex, in hippocampal formation, and in subcortical structures including basal ganglia, thalamus and hypothalamus. Labeling was prominent in limbic areas including olfactory bulb, basal forebrain, pyriform cortex and amygdala. In the mid- and hindbrain, immunolabeled neurons were concentrated in specific nuclei and, in some areas, in specific subregions of those nuclei. Structures of the motor system, including cranial nerve motor nuclei, precerebellar nuclei, the substantia nigra, and the red nucleus were clearly labeled. Staining was intense in cells and/or puncta in the rostral and caudal ventrolateral medullary reticular formation, nucleus tractus solitarius and the caudal vestibular nuclear complex. Within neurons, the ribotide was found predominantly in somata and dendrites; some myelinated axons and occasional synaptic terminals were also immunostained. These data indicate that IAA-RP contributes to the neurochemical phenotype of many neuronal populations and further supports our suggestion that, in autonomic structures, IAA-RP may serve as a chemical mediator in complex circuits involved in blood pressure regulation and, more generally, sympathetic drive.

The neurochemistry of the retina of the larval and postmetamorphic sea lamprey was studied via immunocytochemistry using antibodies directed against the major candidate neurotransmitters [glutamate, glycine, gamma-aminobutyric acid (GABA), aspartate, dopamine, serotonin] and the neurotransmitter-synthesizing enzyme tyrosine hydroxylase. Immunoreactivity to rod opsin and calretinin was also used to distinguish some retinal cells. Two retinal regions are present in larvae: the central retina, with opsin-immunoreactive photoreceptors, and the lateral retina, which lacks photoreceptors and is mainly neuroblastic. We observed calretinin-immunostained ganglion cells in both retinal regions; immunolabeled bipolar cells were detected in the central retina only. Glutamate immunoreactivity was present in photoreceptors, ganglion cells, and bipolar cells. Faint to moderate glycine immunostaining was observed in photoreceptors and some cells of the ganglion cell/inner plexiform layer. No GABA-immunolabeled perikarya were observed. GABA-immunoreactive centrifugal fibers were present in the central and lateral retina. These centrifugal fibers contacted glutamate-immunostained ganglion cells. No aspartate, serotonin, dopamine, or TH immunoreactivity was observed in larvae, whereas these molecules, as well as GABA, glycine, and glutamate, were detected in neurons of the retina of recently transformed lamprey. Immunoreactivity to GABA was observed in outer horizontal cells, some bipolar cells, and numerous amacrine cells, whereas immunoreactivity to glycine was found in amacrine cells and interplexiform cells. Dopamine and serotonin immunoreactivity was found in scattered amacrine cells. Amacrine and horizontal cells did not express classical neurotransmitters (with the possible exception of glycine) during larval life, so transmitter-expressing cells of the larval retina appear to participate only in the vertical processing pathway.

The present immunocytochemical study provides evidence of a previously unrecognized, rich, gamma-aminobutyric acid (GABA)-ergic innervation of the pineal organ in the dogfish (Scyliorhinus canicula). In this elasmobranch, the pineal primordium is initially detected at embryonic stage 24 and grows to form a long pineal tube by stage 28. Glutamic acid decarboxylase (GAD)-immunoreactive (-ir) fibers were first observed at stage 26, and by stage 28, thin GAD-ir fibers were detectable at the base of the pineal neuroepithelium. In pre-hatchling embryos, most fibers gave rise to GAD-ir boutons that were localized in the basal region of the neuroepithelium, although a smaller number of labeled terminals ascended to the pineal lumen. A few pale GAD-ir perikarya were observed within the pineal organ of stage 29 embryos, but GAD-ir perikarya were not observed at other developing stages or in adults. In contrast, GABA immunocytochemistry revealed the presence of GABAergic perikarya and fibers in the pineal organ of late stage embryos and adults. Although high densities of GABAergic cells were observed in the paracommissural pretectum, posterior tubercle, and tegmentum of dogfish embryos (regions previously demonstrated to contain pinealopetal cells), the presence of GABA-ir perikarya in the pineal organ strongly suggests that the rich GABAergic innervation of the elasmobranch pineal organ is intrinsic. This contrasts with the central origin of GABAergic fibers in the pineal gland of some mammals.

We identified the previously unknown structures of ribosylated imidazoleacetic acids in rat, bovine, and human tissues to be imidazole-4-acetic acid-ribotide (IAA-RP) and its metabolite, imidazole-4-acetic acid-riboside. We also found that IAA-RP has physicochemical properties similar to those of an unidentified substance(s) extracted from mammalian tissues that interacts with imidazol(in)e receptors (I-Rs). [“Imidazoline,” by consensus (International Union of Pharmacology), includes imidazole, imidazoline, and related compounds. We demonstrate that the imidazole IAA-RP acts at I-Rs, and because few (if any) imidazolines exist in vivo , we have adopted the term “imidazol(in)e-Rs.”] The latter regulate multiple functions in the CNS and periphery. We now show that IAA-RP ( i ) is present in brain and tissue extracts that exhibit I-R activity; ( ii ) is present in neurons of brainstem areas, including the rostroventrolateral medulla, a region where drugs active at I-Rs are known to modulate blood pressure; ( iii ) is present within synaptosome-enriched fractions of brain where its release is Ca 2+ -dependent, consistent with transmitter function; ( iv ) produces I-R-linked effects in vitro (e.g., arachidonic acid and insulin release) that are blocked by relevant antagonists; and ( v ) produces hypertension when microinjected into the rostroventrolateral medulla. Our data also suggest that IAA-RP may interact with a novel imidazol(in)e-like receptor at this site. We propose that IAA-RP is a neuroregulator acting via I-Rs.

The synapsins are presynaptic membrane-associated proteins involved in neurotransmitter release. They are differentially expressed in tissues and cells of the central and peripheral nervous system. In vestibular end organs of mammals, synapsin I-like immunoreactivity has been reported in efferent and afferent terminals and in afferent nerve calyces surrounding type I hair cells. In addition, synapsin I has recently been described in several non-neural cell lines. The present study was conducted to locate synapsin-like immunoreactivity in the neuronal and non-neuronal cells of the fish crista ampullaris, to examine the possibility that the non-neuronal sensory receptor cells express synapsins in vivo. Synapsin-like immunostaining was visualized by immunofluorescence detection in wholemounts of the toadfish crista ampullaris using multiphoton laser scanning microscopy and by electron microscopic visualization of post-embedding immunogold labeling. The results demonstrate that synapsin-like immunoreactivity is present in vestibular hair cells and efferent boutons of the toadfish crista ampullaris. Afferent endings are not labeled. Staining in hair cells is not associated with the synaptic ribbons, suggesting that there is an additional, non-synaptic role for the synapsins in some non-neuronal cells of vertebrates. Moreover, while the cristae of amniote and anamniote species share many functional attributes, differences in their synaptic vesicle-associated protein profiles appear to reflect their disparate hair cell populations.

The cellular and subcellular localization of L-citrulline was analyzed in the adult rat brain and compared with that of traditional markers for the presence of nitric oxide synthase. Light, transmission electron, and confocal laser scanning microscopy were used to study tissue sections processed for immunocytochemistry employing a monoclonal antibody against L-citrulline or polyclonal anti-neuronal nitric oxide synthase sera, and double immunofluorescence to detect neuronal nitric oxide synthase and L-citrulline co-localization. The results demonstrate that the same CNS regions and cell types are labeled by neuronal nitric oxide synthase polyclonal antisera and L-citrulline monoclonal antibodies, using both immunocytochemistry and immunofluorescence. Short-term pretreatment with a nitric oxide synthase inhibitor reduces L-citrulline immunostaining, but does not affect neuronal nitric oxide synthase immunoreactivity. In the vestibular brainstem, double immunofluorescence studies show that many, but not all, neuronal nitric oxide synthase-positive cells co-express L-citrulline, and that local intracellular patches of intense L-citrulline accumulation are present in some neurons. Conversely, all L-citrulline-labeled neurons co-express neuronal nitric oxide synthase. Cells expressing neuronal nitric oxide synthase alone are interpreted as neurons with the potential to produce nitric oxide under other stimulus conditions, and the subcellular foci of enhanced L-citrulline staining are viewed as intracellular sites of nitric oxide production. This interpretation is supported by ultrastructural observations of subcellular foci with enhanced L-citrulline and/or neuronal nitric oxide synthase staining that are located primarily at postsynaptic densities and portions of the endoplasmic reticulum. We conclude that nitric oxide is produced and released at focal sites within neurons that are identifiable using L-citrulline as a marker. Copyright 2002 IBRO.

Deaggregation, the partial reversal of the initial aggregation of platelets is observed following low, but not higher, micromolar ADP concentrations. This study tested the hypothesis that deaggregation results from a balance between concurrent, opposing, aggregation and deaggregation processes which are ADP (adenosine 5'-diphosphate) receptor occupancy-dependent. Aggregation of human platelet-rich plasma (PRP) prepared in r-hirudin was assayed in a 96-well plate reader over 20 min by measurement of the optical density (OD) at 580 nm. Aggregation and the time to reach peak aggregation were directly proportional to ADP receptor occupancy. The magnitude and time course of the response to ADP were comparable to those previously reported with standard aggregometry. The rate constant of platelet deaggregation, as assessed by a four-compartment kinetic model, was inversely proportional to agonist concentration. The ratio of the rate constants of aggregation and deaggregation was receptor occupancy-dependent and directly proportional to aggregation. Consequently, platelet aggregation was proportional, and deaggregation inversely proportional, to ADP receptor occupancy. We propose that the response of PRP to ADP and to 2-MeS-ADP (2-methylthioadenosine-diphosphate), in vitro, consists of at least two active, concurrent processes, aggregation and deaggregation. Incremental occupancy of the P2T ADP receptor subtype attenuates deaggregation and governs the balance between these two processes.

The angular vestibulo-ocular reflex maintains gaze during head movements. It is thought to be mediated by two components: direct and velocity storage pathways. The direct angular vestibulo-ocular reflex is conveyed by a three neuron chain from the labyrinth to the ocular motoneurons. The indirect pathway involves a more complex neural network that utilizes a portion of the vestibular commissure. The purpose of the present study was to identify the ultrastructural characteristics of commissural neurons in the medial vestibular nucleus that are related to the velocity storage component of the angular vestibulo-ocular reflex. Ultrastructural studies of degenerating medial vestibular nucleus neurons were conducted in monkeys following midline section of rostral medullary commissural fibers with subsequent behavioral testing. After this lesion, oculomotor and vestibular functions attributable to velocity storage were abolished, whereas the direct angular vestibulo-ocular reflex pathway remained intact. Since this damage was functionally discrete, degenerating neurons were interpreted as potential participants in the velocity storage network. Ultrastructural observations indicate that commissural neurons related to velocity storage are small and medium sized cells having large nuclei with deep indentations and relatively little cytoplasm, which are located in the lateral crescents of rostral medial vestibular nucleus. The morphology of degenerating dendritic profiles varied. Some contained numerous round or tubular mitochondria in a pale cytoplasmic matrix with few other organelles, while others had few mitochondria but many cisterns and vacuoles in dense granular cytoplasm. The commissural nature of these cells was further suggested by the presence of two different types of degenerating axon terminals in the rostral medial vestibular nucleus: those with a moderate density of large spherical synaptic vesicles, and those with pleomorphic, primarily ellipsoid synaptic vesicles. The recognition of two types of degenerating terminals further supports our interpretation that at least two morphological types of commissural neurons participate in the velocity storage network. The degenerating boutons formed contacts with a variety of postsynaptic partners. In particular, synapses were observed between degenerating boutons and non-degenerating dendrites, and between intact terminals and degenerating dendrites. However, degenerating pre- and postsynaptic elements were rarely observed in direct contact, suggesting that additional neurons are interposed in the indirect pathway commissural system. On the basis of these ultrastructural observations, it is concluded that vestibular commissural neurons involved in the mediation of velocity storage have distinguishing ultrastructural features and synaptology, that are different from those of direct pathway neurons.

The effects of a subtherapeutic regimen of rapamycin (RAPA) combined with CsA and donor-specific blood transfusions (DST) were investigated in the ACI to LEW cardiac allograft model. Three protocols were studied. In protocol 1, recipients received a DST (day -1), RAPA intravenously at 0.08 mg/kg/day (day -1 to +5), and CsA intramuscularly at 5 mg/kg/day (day -1 to +1). In protocol 2, the course of CsA was extended to day +5, and in protocol 3 the treatment was further modified to include a second DST on day +3. Control groups received drug treatments alone or combined with DST or third-party blood transfusions. In all protocols, RAPA, CsA, or DSTs alone had little or no effect on graft survival. RAPA-DST or CsA-DST also had no effects beyond the ones induced by the drugs alone. In the RAPA-CsA groups, graft survivals were similar to the ones obtained with CsA alone. In contrast, in each of the protocols, the RAPA-CsA-DST treatment resulted in significant prolongation of graft survival as compared with all controls (P < 0.05). The administration of a second DST (CsA-RAPA-DSTx2) provided no additional benefits over a single DST. Lymph node cells from CsA-RAPA-DST subjects were hyporesponsive in MLR against donor cells and suppressed the proliferation of normal LEW cells in an MLR coculture assay, suggesting the presence of suppressor cells. Furthermore, in the CsA-RAPA-DST group, the anti-donor antibody response was suppressed. These data demonstrate that RAPA, CsA, and DST interact positively by inducing a clear-cut and significant prolongation of allograft survival in the rat model under conditions in which the individual components of the treatment are essentially ineffectual.

This study was performed to determine the correlation of tumor ras and c-myc oncogene expression with clinical and prognostic variables in patients prone to develop colorectal cancer. One hundred eighteen patients with colorectal cancer were studied; mean age was 40 years. Fifty-three were young patients (age 40 or less), 49 had ulcerative colitis, and 16 had multiple polyposis coli. Immunoperoxidase stains of paraffin-embedded cancer sections were performed for the c-myc and ras proteins. ras staining was found to correlate with Dukes stage and prognosis. Patients with tumors negative for ras protein stain had an actuarial five-year survival of 61 percent versus 44 percent for those tumors with a positive stain (P less than 0.05). This correlation was not seen with the c-myc stain. Positive ras oncogene stain appears to be a useful indicator of advanced stage and poor prognosis in colorectal cancer occurring in cancer-prone patients.

Sixteen patients with polyposis coli and cancer were studied retrospectively to determine the incidence of DNA ploidy abnormalities in the tumors and synchronous polyps. Six patients (37 percent) had nondiploid tumors. Nondiploid tumors were more likely to be advanced and had a significantly worse prognosis (17 percent vs. 76 percent 5-year survival; P less than 0.01). Only 4 of 20 polyps studied were nondiploid. There was no association between tumor and polyp ploidy. All nondiploid polyps were found in patients with synchronous diploid cancers. Patients with nondiploid polyps were more likely to be older and have more advanced tumors than those with diploid polyps. DNA ploidy abnormalities seem to occur with the same frequency in polyposis coli as in the nonpolyposis population, and tumor ploidy correlates with prognosis.

Several studies have shown that the presence of DNA content abnormalities, measured by flow cytometry, may correlate with a poor prognosis in a variety of cancers. The predictive value of DNA content in patients with small rectal cancers has not been well determined. Thirty-nine patients with primary rectal adenocarcinoma smaller than 3 cm were studied in a comparison of DNA content with established prognostic variables. The following covariates were evaluated for their prognostic value: sex, age, tumor size, location, distal margin, Dukes' classification, tumor differentiation, and DNA content. DNA content was assessed by flow cytometric analysis, and each tumor was categorized as diploid or nondiploid. Of the parameters studied, Dukes' classification and tumor DNA content were found to be independent prognostic indicators. Determination of DNA content seems to provide additional useful prognostic information in patients with small rectal tumors.

The performance of a new dialysis prosthesis designed to self-seal after puncture was tested ex vivo and in vivo. It consists of two coaxial polytetrafluoroethylene tubes (PTFE), the space between them filled with silicone rubber sealant (PTFE-sil). Ex vivo: Three PTFE-sil, three double PTFE (without silicone), and three single PTFE grafts were placed sequentially between scribner shunts in the hind limb of four dogs. Bleeding on puncturing with an 18-gauge needle was measured for 30 seconds. PTFE-sil bled less than the controls (g): PTFE-sil; 16 +/- 18; double PTFE: 32 +/- 10; single PTFE: 52 +/- 19 (p less than 0.001). In vivo: Six PTFE-sil and five single PTFE grafts were interposed between the carotid artery and jugular vein of dogs and were punctured with a 16-gauge needle on days 1, 3, and 7. Bleeding was measured through an incision over the puncture site at 5 minutes. In 11 punctures of PTFE-sil, there was no bleeding; three bled less than 20 g. In 13 control punctures, none bled less than 70 g. Patency: Grafts were studied for patency in arteriovenous (AV) fistulas in 34 dogs. Each dog received a PTFE-sil graft in one groin and a single PTFE control graft in the other. At 4 months, patency rates were: PTFE-sil, 84%; single PTFE, 87% (NS). Four months after implantation, hemostasis after puncture in PTFE-sil grafts required 70 +/- 49 seconds versus 207 +/- 48 seconds for PTFE grafts (p less than 0.005). In conclusion, PTFE-sil grafts are self-sealing, can be used immediately after implantation, and need minimal compression after needle removal.

The immunosuppressive activity of CVF was evaluated in mice immunized with sheep erythrocytes and dinitrophenylated proteins. Serum antibody levels to these immunogens were estimated in activity units and on a weight basis for IgG. IgM as well as IgG antibody responses were diminished in mice pretreated with CVF. However when soluble immunogens were incorporated in CFA the suppressive effect associated with CVF was inapparent. It is suggested that C depletion per se may not fully account for the observed immunosuppression. The latter may result not only from the depression of C3 levels but also from the biologic activities of C cleavage products some of which modulate the secretory functions and state of activation of macrophages.

We investigated the effect of pretransplant conditioning as a way to reduce the need for the aggressive immunosuppressive therapy reportedly required in small bowel (SB) allograft recipients. LEW rats were conditioned with (1) a donor-specific blood transfusion (DST) on day -8 and a concurrent 5-day course of CsA (10 mg/kg/day); (2) a nonspecific blood transfusion and CsA; (3) CsA alone. A 10-cm segment of the host native bowel was then replaced with an equivalent segment of SB obtained from ACI rats. Postoperative treatment consisted of CsA at 2.5 mg/kg/day for 30 days. Rats conditioned with a nonspecific transfusion and CsA or with CsA alone survived for 14.1 +/- 5.8 and 18.3 +/- 5.7 days, respectively. In contrast, rats conditioned with DST and CsA survived for 60.3 +/- 36.2 days (P less than 0.001 vs. both controls). Biopsies taken from long-term survivors showed a normal bowel architecture. The function of the allografts was studied in a group of animals totally deprived of their native bowel and transplanted with a 30-cm segment of ACI SB. CsA-DST-treated recipients survived an average of 90 +/- 43 days and grew at a rate comparable to isografted animals. Treated allograft recipients had maltose absorption indistinguishable from isografted controls at all times tested. In contrast, maltose absorption was severely impaired in recipients rejecting their grafts. This study demonstrates that long-term survival of SB allograft recipients can be achieved with good functional results with low doses of CsA in recipients conditioned with DST and CsA.

A self-sealing polytetrafluoroethelene (PTFE)-silicone graft designed to be used early after implantation was placed in 30 end-stage renal disease patients. Thirty-five conventional PTFE grafts were used in the control group. All patients were followed for 1 year. In the PTFE-silicone graft group, median time for first use was 1 day after implantation. Conventional PTFE grafts were first used 2 to 4 weeks after implantation. Early punctures of the PTFE-silicone grafts (first 10 sessions), although more difficult to perform than late punctures, were not more difficult than punctures of conventional PTFE grafts. Late punctures in PTFE-silicone grafts were easier than conventional PTFE graft punctures. Bleeding after needle removal was significantly decreased after early and late punctures of PTFE-silicone grafts compared with conventional PTFE grafts (p less than 0.001). The PTFE-silicone graft can be used immediately after implantation, sparing patients additional access procedures for short-term dialysis. This results in less morbidity, decreased bleeding complications, shorter hospital stay, and a significant reduction in expenses.