Judith A. Neubauer

Blood O2 saturation determination in frozen tissue

Abstract We have developed a method for microspectrophotometric determination of oxygen saturation in small arteries and veins of a quick-frozen tissue. Optical densities of 10- to 30-μm sections of frozen dog blood were determined with a Zeiss microspectrophotometer, and oxygen saturation was determined by a three-wavelength (560, 523, 506 nm) method. Blood sections were covered with silicone oil to prevent O 2 diffusion. Calibration lines were constructed by comparing absorbance ratios computed by the three-wavelength method against blood O 2 saturation determined by the Van Slyke method. The method was tested in arteries and veins of frozen dog gracilis muscle against Van Slyke. The mean saturation determined from three different vessels had an accuracy of ±2.6%.

Carbonic Anhydrase and Sensory Function in the Central Nervous System

The carbonic anhydrase (CA) (EC 4.2.1.1.) isozymes catalyze the reversible hydration of CO2. CA activity is present in the central nervous system (CNS).1,10 In brain tissue, the high-activity isozyme, presence of CA II, and the membrane- bound isozyme, CA IV, has been demonstrated.4a,8,33

Heme oxygenase is necessary for the excitatory response of cultured neonatal rat rostral ventrolateral medulla neurons to hypoxia

Heme oxygenase has been linked to the oxygen-sensing function of the carotid body, pulmonary vasculature, cerebral vasculature, and airway smooth muscle. We have shown previously that the cardiorespiratory regions of the rostral ventrolateral medulla are excited by local hypoxia and that heme oxygenase-2 (HO-2) is expressed in the hypoxia-chemosensitive regions of the rostral ventrolateral medulla (RVLM), the respiratory pre-Bötzinger complex, and C1 sympathoexcitatory region. To determine whether heme oxygenase is necessary for the hypoxic-excitation of dissociated RVLM neurons (P1) cultured on confluent medullary astrocytes (P5), we examined their electrophysiological responses to hypoxia (NaCN and low Po(2)) using the whole-cell perforated patch clamp technique before and after blocking heme oxygenase with tin protoporphyrin-IX (SnPP-IX). Following the electrophysiological recording, immunocytochemistry was performed on the recorded neuron to correlate the electrophysiological response to hypoxia with the expression of HO-2. We found that the responses to NaCN and hypoxia were similar. RVLM neurons responded to NaCN and low Po(2) with either depolarization or hyperpolarization and SnPP-IX blocked the depolarization response of hypoxia-excited neurons to both NaCN and low Po(2) but had no effect on the hyperpolarization response of hypoxia-depressed neurons. Consistent with this observation, HO-2 expression was present only in the hypoxia-excited neurons. We conclude that RVLM neurons are excited by hypoxia via a heme oxygenase-dependent mechanism.

Heme oxygenase-1 and chronic hypoxia.

A myriad of changes are necessary to adapt to chronic hypoxemia. Key among these changes increases in arterial oxygen carrying capacity, ventilation and sympathetic activity. This requires the induction of several gene products many of which are regulated by the activity of HIF-1α, including HO-1. Induction of HO-1 during chronic hypoxia is necessary for the continued breakdown of heme for the enhanced production of hemoglobin and the increased respiratory and sympathetic responses. Several human HO-1 polymorphisms have been identified that can affect the expression or activity of HO-1. Associations between these polymorphisms and the prevalence of hypertension have recently been assessed in specific populations. There are major gaps in our understanding of the mechanisms of how HO-1 mediates changes in the activity of the hypoxia-sensitive chemosensors and whether HO-1 polymorphisms are an important factor in the integrated response to chronic hypoxia. Understanding how HO-1 mediates cardiorespiratory responses could provide important insights into clinical syndromes such as obstructive sleep apnea.

Opioids protect against substantia nigra cell degeneration under conditions of iron deprivation: A mechanism of possible relevance to the Restless Legs Syndrome (RLS) and Parkinson's Disease

Hypofunction of the endogenous opioid, dopamine and iron systems are implicated in the pathogenesis of Restless Legs Syndrome (RLS). Therefore, we probed the interrelationship of these 3 systems in an in vitro model. Cell cultures of the substantia nigra (SN) of Sprague-Dawley rats were established and the cells were determined to be primarily dopaminergic. The numbers of cells surviving under different concentrations of the iron chelator desferoxamine were reduced in a concentration and time dependent manner (p<0.01 at day 10, n=19). The cell death was determined to be apoptotic and DNA analysis revealed that 48-hour 100 μM desferoxamine exposure caused DNA fragmentation of the cells. Pre-administration of the δ-opioid peptide [D-Ala2, D-Leu5]Enkephalin (DADLE) significantly protected the SN cells from damage by iron deficiency (n=6, p<0.01). Our previous studies indicate that the DNA-damage induced apoptosis family gene P53 is activated in this model and that pre-exposure to DADLE prevents this activation. The implications of this model are that in RLS patients with iron deficiency, dopaminergic system dysfunction may result and an intact endogenous opioid system or opioid treatment may protect the dopamine system from dysfunction. Implications of this model for Parkinsons Disease are also briefly discussed.

Medical student research exposure via a series of modular research programs

Background The falling percentage of doctors of medicine applying for National Institute of Health-funded research grants is 1 indicator that physician-scientists are a disappearing breed. This is occurring at a time when increased translational, disease-oriented, patient-oriented, and clinical research are national goals. One of the keys to providing sufficient numbers of physician-scientists to support this goal is the active targeting of medical students. We hypothesize that an improved research program infrastructure and responsiveness to changing student needs will increase student participation in research-oriented electives. Methods We have developed a student research program consisting of 2 Students Interested in Research noncredit electives (lecture and laboratory based), summer fellowships, support for year-out fellowships, and a Distinction in Research program that spans undergraduate medical education. Student participation and short-term research outcomes from fall 2004 through spring 2008 are analyzed to examine program efficacy. Results Students involved in the early parts of the program initially experienced higher application and success rates for summer funding opportunities, but as the program has matured, these rates have fallen in line with the class average. Independently, students participating in later portions of the program increasingly submit or publish a first author paper and have taken a year off for research during medical school. Overlap of participation in the programs is generally smaller than expected. Conclusion Although structured programs can provide step-wise research experiences of increasing intensity, students may not experience a training pipeline in which each stage relies on those before and after, and instead may sample an a la carte selection of research-based enrichment opportunities.

Oxygen saturation determination in frozen blood

Abstract A spectroscopic method for O 2 saturation determination in frozen blood has been developed. In the 500–575 nm range, frozen oxygenated and deoxygenated blood had essentially the same spectral absorption character as liquid blood, although apparent absorbance was much higher than liquid blood. The standard two-wavelength method for spectroscopic determination of O 2 saturation of blood was inadequate to correct for the nonspecific light loss through the ice crystals. A method for O 2 saturation determination using three wavelength (506, 523, and 560 nm) measurements is reported here. Compared with blood O 2 saturation determined by the Van Slyke technique, this three-wavelength method demonstrated an accuracy of 2–5% within 95% confidence limits. It also compared favorably with a previously reported four-wavelength geometric computational technique. The rate of freezing did not influence the absorption spectra nor the percentage saturation determination. Samples could be stored at −25° up to 49 days with no change in O 2 saturation. Our calculations reveal that the upper limit of blood O 2 saturation increase in a closed system due to freezing is 2.3%. The upper limits of variation in blood O 2 saturation due to freezing blood with various hemoglobin concentrations or at various initial pH was calculated to be about 1% or less. This method may be adapted for microspectrophotometric determination of regional arteriovenous blood O 2 saturation difference in quick frozen tissue.

Heme oxygenase-1-dependent central cardiorespiratory adaptations to chronic hypoxia in mice

Adaptations to chronic hypoxia (CH) could reflect cellular changes within the cardiorespiratory regions of the rostral ventrolateral medulla (RVLM), the C1 region, and the pre-Bötzinger complex (pre-BötC). Previous studies have shown that the hypoxic chemosensitivity of these regions are heme oxygenase (HO) dependent and that CH induces HO-1. To determine the time course of HO-1 induction within these regions and explore its relevance to the respiratory and sympathetic responses during CH, the expression of HO-1 mRNA and protein in the RVLM and measures of respiration, sigh frequency, and sympathetic activity (spectral analysis of heart rate) were examined during 10 days of CH. Respiratory and sympathetic responses to acute hypoxia were obtained in chronically instrumented awake wild-type (WT) and HO-1 null mice. After 4 days of CH, there was a significant induction of HO-1 within the C1 region and pre-BötC. WT mice acclimated to CH by increasing peak diaphragm EMG after 10 days of CH but had no change in the respiratory response to acute hypoxia. There were no significant differences between WT and HO-1 null mice. In WT mice, hypoxic sigh frequency and hypoxic sensitivity of sympathetic activity initially declined before returning toward baseline after 5 days of CH, correlating with the induction of HO-1. In contrast, HO-1 null mice had a persistent decline in hypoxic sigh frequency and hypoxic sensitivity of sympathetic activity. We conclude that induction of HO-1 in these RVLM cardiorespiratory regions may be important for the hypoxic sensitivity of sighs and sympathetic activity during CH.

Heme oxygenase-1 Dependent Central Cardiorespiratory Adaptations to Chronic Intermittent Hypoxia in Mice.

Chronic intermittent hypoxia (CIH) increases sympathetic tone and respiratory instability. Our previous work showed that chronic hypoxia induces the oxygen-sensing enzyme heme oxygenase-1 (HO-1) within the C1 sympathoexcitatory region and the pre-Bötzinger complex (pre-BötC). We therefore examined the effect of CIH on time course of induced expression of HO-1 within these regions and determined whether the induction of HO-1 correlated with changes in respiratory, sigh frequency, and sympathetic responses (spectral analysis of heart rate) to acute hypoxia (10% O2) during 10 days of exposure to CIH in chronically instrumented awake wild-type (WT) and HO-1 null mice (HO-1-/-). HO-1 was induced within the C1 and pre-BötC regions after 1 day of CIH. There were no significant differences in the baseline respiratory parameters between WT and HO-1-/- Prior to CIH, acute hypoxia increased respiratory frequency in both WT and HO-1-/-; however, minute diaphragm electromyogram activity increased in WT but not HO-1-/- The hypoxic respiratory response after 1 and 10 days of CIH was restored in HO-1-/- CIH resulted in an initial significant decline in 1) the hypoxic sigh frequency response, which was restored in WT but not HO-1-/-, and 2) the baseline sympathetic activity in WT and HO-1-/-, which remained stable subsequently in WT but not in HO-1-/- We conclude that 1) CIH induces expression of HO-1 in the C1 and pre-BötC regions within 1 day and 2) HO-1 is necessary for hypoxia respiratory response and contributes to the maintenance of the hypoxic sigh responses and baseline sympathetic activity during CIH.

Central Adaptation to Hypoxia

It is well established that in anesthetized animals, in the absence of peripheral chemoreceptor stimulation, hypoxia produces a depression of breathing. Recently, there has been considerable interest in discerning the mechanisms responsible for the central depression of respiration during hypoxia. A basic issue that arises when considering the mechanisms responsible for hypoxic modulation of central respiratory output is whether respiratory neuronal activity is simply limited by substrate availability within the brain, or whether this depression represents an active inhibition of neuronal activity. Such inhibition could serve to minimize energy use during hypoxia by limiting motor activity as well as conserving high energy substrates that would be used normally to reestablish transmembrane ionic gradients dissipated during neuronal activity. Thus, neuronal inhibition might subserve a protective function by affording tolerance to hypoxic environments. This ability to “down-regulate” metabolic activity during hypoxia is a well-characterized phenomenon in many lower vertebrates, for example, the diving turtle (46), but may also be physiologically relevant in both unanesthetized neonatal and adult mammals.