Miljan R. Stankovic

Non-invasive optical monitoring of the newborn piglet brain using continuous-wave and frequency-domain spectroscopy

We have used continuous-wave (CW) and frequency-domain spectroscopy to investigate the optical properties of the newborn piglet brain in vivo and non-invasively. Three anaesthetized, intubated, ventilated and instrumented newborn piglets were placed into a stereotaxic instrument for optimal experimental stability, reproducible probe-to-scalp optical contact and 3D adjustment of the optical probe. By measuring the absolute values of the brain absorption and reduced scattering coefficients at two wavelengths (758 and 830 nm), frequency-domain spectroscopy provided absolute readings (in contrast to the relative readings of CW spectroscopy) of cerebral haemoglobin concentration and saturation during experimentally induced perturbations in cerebral haemodynamics and oxygenation. Such perturbations included a modulation of the inspired oxygen concentration, transient brain asphyxia, carotid artery occlusion and terminal brain asphyxia. The baseline cerebral haemoglobin saturation and concentration, measured with frequency-domain spectroscopy, were about 60% and 42 microM respectively. The cerebral saturation values ranged from a minimum of 17% (during transient brain asphyxia) to a maximum of 80% (during recovery from transient brain asphyxia). To analyse the CW optical data, we have (a) derived a mathematical relationship between the cerebral optical properties and the differential pathlength factor and (b) introduced a method based on the spatial dependence of the detected intensity (dc slope method). The analysis of the cerebral optical signals associated with the arterial pulse and with respiration demonstrates that motion artefacts can significantly affect the intensity recorded from a single optode pair. Motion artefacts can be strongly reduced by combining data from multiple optodes to provide relative readings in the dc slope method. We also report significant biphasic changes (initial decrease and successive increase) in the reduced scattering coefficient measured in the brain after the piglet had been sacrificed.

Streptozotocin-induced diabetes in the rat is associated with changes in vaginal hemodynamics, morphology and biochemical markers

BackgroundDiabetes is associated with declining sexual function in women. However, the effects of diabetes on genital tissue structure, innervation and function remains poorly characterized. In control and streptozotocin-treated female rats, we investigated the effects of diabetes on vaginal blood flow, tissue morphology, and expression of arginase I, endothelial nitric oxide synthase (eNOS) and cGMP-dependent protein kinase (PKG), key enzymes that regulate smooth muscle relaxation. We further related these changes with estrogen receptor alpha (ERα) and androgen receptor (AR) expression.ResultsIn addition to significantly elevated blood glucose levels, diabetic rats had decreased mean body weight, lower levels of plasma estradiol, and higher plasma testosterone concentration, compared to age-matched controls. Eight weeks after administration of buffer (control) or 65 mg/kg of streptozotocin (diabetic), the vaginal blood flow response to pelvic nerve stimulation was significantly reduced in diabetic rats. Histological examination of vaginal tissue from diabetic animals showed reduced epithelial thickness and atrophy of the muscularis layer. Diabetic animals also had reduced vaginal levels of eNOS and arginase I, but elevated levels of PKG, as assessed by Western blot analyses. These alterations were accompanied by a reduction in both ERα and AR in nuclear extracts of vaginal tissue from diabetic animals.ConclusionIn ovariectomized (estrogen deficient) animals, previous reports from our lab and others have documented changes in blood flow, tissue structure, ERα, arginase I and eNOS that parallel those observed in diabetic rats. We hypothesize that diabetes may lead to multiple disruptions in sex steroid hormone synthesis, metabolism and action. These pathological events may cause dramatic changes in tissue structure and key enzymes that regulate cell growth and smooth muscle contractility, ultimately affecting the genital response during sexual arousal.

Testosterone Increases Blood Flow and Expression of Androgen and Estrogen Receptors in the Rat Vagina

INTRODUCTION The mechanisms by which testosterone modulates female genital sexual arousal responses are poorly understood. AIM To investigate the effects of testosterone on vaginal blood flow and the expression of estrogen and androgen receptor proteins in the rat vagina. METHODS Mature female Sprague-Dawley rats were sham-operated (intact) or ovariectomized. Fourteen days after ovariectomy, animals were continuously infused with vehicle or varying doses of testosterone (5.5-55 microg/day). After 2 weeks of treatment, vaginal blood flow in response to pelvic nerve stimulation was measured by laser Doppler flowmetry. Plasma levels of testosterone and estradiol were determined by radioimmunoassay and epithelial thickness was examined in fixed vaginal tissue sections. Androgen and estrogen receptor levels were assessed by equilibrium radioligand binding and by Western blot analyses. RESULTS Vaginal blood flow responses were significantly reduced in ovariectomized rats and normalized in animals infused with testosterone. Ovariectomy increased the expression of estrogen receptors and reduced the expression of androgen receptors with no change in receptor-ligand affinity. Testosterone increased the expression of both androgen and estrogen receptors in the vagina. While physiological (11 microg/day) and supraphysiological (55 microg/day) concentrations of testosterone normalized vaginal tissue weight, uterine tissue and whole body weights were not significantly different from ovariectomized rats infused with vehicle. Testosterone infusion, even at supraphysiological concentrations, did not change plasma estradiol levels when compared to vehicle-infused, ovariectomized rats. Likewise, the vaginal epithelium of testosterone-infused rats remained atrophic, similar to vehicle-infused, ovariectomized rats, indicating that testosterone is not aromatized to estrogens at significant levels in the vagina. CONCLUSIONS Our data suggest that testosterone regulates androgen and estrogen receptor protein expression in the vagina and enhances vaginal perfusion by an androgen-dependent mechanism. We conclude that testosterone plays an important role in modulating the physiology of the vagina and contributes to improvement of genital sexual arousal responses.

Non-invasive optical mapping of the piglet brain in real time

We have performed non-invasive, real-time optical mapping of the piglet brain during a subcortical injection of autologous blood. The time resolution of the optical maps is 192 ms, thus allowing us to generate a real-time video of the growing subcortical hematoma. The increased absorption at the site of blood injection is accompanied by a decreased absorption at the contralateral brain side. This contralateral decrease in the optical absorption and the corresponding time traces of the cerebral hemoglobin parameters are consistent with a reduced cerebral blood flow caused by the increased intracranial pressure.

Hemodynamic Evaluation of the Female Sexual Arousal Response in an Animal Model

The goal of this study was to assess the utility of existing and new techniques for characterizing and measuring hemodynamic changes in the vagina and clitoris in response to pelvic nerve stimulation (PNS) in an animal model. Using female New Zealand White rabbits, we measured the following parameters before, during, and after PNS at 4, 16, and 32 hertz (Hz): clitoral hemoglobin (Hb) content by laser oximetry, clitoral blood flow by laser Doppler flowmetry, vaginal luminal pressure of upper and lower segments, and clitoral intracavernosal pressure. Clitoral tissue concentrations of total and oxygenated hemoglobin (oxy-Hb) increased in a frequency-dependent manner while deoxygenated hemoglobin (deoxy-Hb) concentration decreased. The duration of the responses at 16 and 32 Hz were significantly greater than at 4 Hz. Clitoral blood flow increased significantly only at 32 Hz with a prolonged response duration, relative to 4 Hz. PNS caused vaginal luminal pressure changes that were highly variable, but qualitatively different, between the upper and lower regions. Clitoral intracavernosal pressure did not change significantly in response to PNS. Measurement of changes in tissue Hb content by the novel technique of laser oximetry provides a direct assessment of blood flow in a noninvasive manner and may prove to be a powerful tool in evaluating hemodynamic aspects of the female genital sexual response.

Effects of tamoxifen on vaginal blood flow and epithelial morphology in the rat

BackgroundTamoxifen, a selective estrogen receptor modulator with both estrogenic and anti-estrogenic activity, is widely used as adjuvant therapy in breast cancer patients. Treatment with tamoxifen is associated with sexual side effects, such as increased vaginal dryness and pain/discomfort during sexual activity. There have been limited investigations of the effect of tamoxifen on estrogen-dependent peripheral genital arousal responses. The objective of this study was to investigate the effects of tamoxifen on vaginal physiology in the rat.MethodsFemale Sprague-Dawley rats were subjected to sham surgery or bilateral ovariectomy. After 2 weeks, sham-operated rats were implanted with subcutaneous osmotic infusion pumps containing vehicle (control) or tamoxifen (150 μg/day). Ovariectomized rats were similarly infused with vehicle. After an additional 2 weeks, vaginal blood flow responses to pelvic nerve stimulation were measured by laser Doppler flowmetry and vaginal tissue was collected for histological and biochemical assay.ResultsTamoxifen treatment did not change plasma estradiol concentrations relative to control animals, while ovariectomized rats exhibited a 60% decrease in plasma estradiol. Tamoxifen treatment caused a significant decrease in mean uterine weight, but did not alter mean vaginal weight. Vaginal blood flow was significantly decreased in tamoxifen-infused rats compared to controls. Similar to ovariectomized animals, estrogen receptor binding was increased and arginase enzyme activity was decreased in tamoxifen-infused rats. However, different from control and ovariectomized animals, the vaginal epithelium in tamoxifen-infused rats appeared highly mucified. Periodic acid-Schiff staining confirmed a greater production of carbohydrate-rich compounds (e.g. mucin, glycogen) by the vaginal epithelium of tamoxifen-infused rats.ConclusionThe observations suggest that tamoxifen exerts both anti-estrogenic and pro-estrogenic effects in the vagina. These physiological alterations may eventually lead to vaginal atrophy and compromise sexual function.

Real-time optical imaging of experimental brain ischemia and hemorrhage in neonatal piglets.

Abstract Our objective was to study the development of experimental brain ischemia and hemorrhage by real-time optical imaging. Optical imaging is based on the ability of near infrared light to non-invasively penetrate through the intact scalp and skull and measure brain concentrations of oxy- and deoxyhemoglobin, dominant brain absorbers. Optical imaging was performed in 7 anesthetized, instrumented, and ventilated newborn piglets subjected to the injection of 0.3 cc of saline followed by 2 cc of blood into the left frontal subcortical brain region via a needle inserted through the skull with stereotactic guidance. The image-acquisition rate of 5.26 images per sec allowed for real-time imaging. The detection threshold of the imager at the estimated depth of 1–1.5 cm was ∼, 70 μL for saline and ∼,40 μL for blood. The imager readily detected five subcortical hematomas and two large bilateral subarachnoid hemorrhages. The imager detected a global decrease in brain absorption associated with the volume-injection-related increase in intracranial pressure in the surrounding ipsi- lateral and contralateral brain. Any decrease in brain absorption is an equivalent to brain ischemia. This study demonstrates the capability of optical imaging in detecting brain ischemia and hemorrhage in real-time with high temporal and spatial resolution.

Role of frequency domain optical spectroscopy in the detection of neonatal brain hemorrhage — A newborn piglet study

OBJECTIVE Inability of continuous wave (CW) optical spectroscopy to measure changes in scattering, and the use of an arbitrary rather than an actual baseline, makes the CW method highly susceptible to errors that can lead to a false-positive or false-negative diagnosis. Our objective was to assess whether, and to what extent, the use of quantitative frequency domain spectroscopy would improve our ability to detect and monitor the development of brain hemorrhage. METHODS A dual-channel frequency-domain tissue spectrometer (Model 96208, ISS, Inc., Champaign, IL) was used to monitor the development of experimental subcortical and periventricular-intraventricular hemorrhage (IVH) in 10 newborn piglets (blood injection model). The multidistance approach was employed to calculate the absorption and reduced scattering coefficients and hemoglobin changes from the ac, dc, and phase values acquired at four different source-detector distances and at 752 nm and 830 nm. RESULTS There were significant absorption and scattering changes in the subcortical hematoma (n = 5) and the IVH groups (n = 5). The smallest detectable amount of blood in the brain was 0.04 ml. Changes associated with subcortical hematoma were several times higher than those associated with IVH, and correlated better with the estimated cross-sectional area of the hematoma than with the volume of the injected blood. As opposed to IVH, there was a significant absorption difference between the injured (subcortical hematoma) and normal side of the brain, probably because in case of IVH a significant volume of the injected blood had accumulated/spread beyond the reach of the probe. CONCLUSION Clearly, frequency-domain spectroscopy cannot increase our ability to quantify the volume (size) or the oxygenation of the injected blood, especially in the case of IVH. However, the ability to quantify the baseline tissue absorption and scattering would significantly improve diagnostic performance, and may allow for early identification and treatment of neonatal brain hemorrhage.

First RealTime Optical Imaging of Experimental Brain Ischemia and Hemorrhage in Neonatal Piglets

The American Pediatric Society and The Society for Pediatric Research 1999 Abstract Pediatric Research (1999) 45, 347A–347A; doi:10.1203/00006450-199904020-02066 First RealTime Optical Imaging of Experimental Brain Ischemia and Hemorrhage in Neonatal Piglets Miljan R Stankovic 1 , Dev Maulik 1 , Warren Rosenfeld 1 , Phillip G Stubblefield 1 , Maria Angela Franceschini 1 , Dennis Hueber 1 , Enrico Gratton 1 and Sergio Fantini 1 1 Winthrop Univ. Hospital, Mineola, NY, Boston Univ., Boston, MA, Dept Physics, Urbana, IL, ISS, Champaign, IL Abstract 2050 Poster Session II, Sunday, 5/2 (poster 229) Optical imaging, based on the ability of near infrared light to non-invasively penetrate through the intact scalp and skull, can monitor cerebral hemodynamics and oxygenation in real-time, assuming that images are acquired fast enough. Previously reported acquisition times of 5 s, 10 min, 2 h, or even 1 to 3 days per image, were far too slow to allow for real-time imaging, particularly in clinical settings where acute events are the focus of our attention. This study presents the first application of real-time optical imaging in the detection of experimental brain ischemia and hemorrhage and the accompanying cerebrovascular changes in newborn piglets. Optical imaging was performed on 7 anesthetized, instrumented, and ventilated newborn piglets secured within a stereotaxic instrument. We used a modified near-infrared oximeter and a custom-built 16 - light - source - 2 - detector optical probe rendering 64 pixel / 4 × 4 cm / 2-D brain absorption images with the acquisition time of 0.19 s per image (or 5.26 images per sec). Once the baseline was recorded (Fig 1), 0.3 cc of saline and 2 cc of blood were injected into the left frontal subcortical brain region aimed to induced focal and global anatomical and functional brain absorption changes. The imager readily detected: 1) focal changes in brain absorption caused by the appearance of the saline (white changes) and blood (dark changes) in the brain featuring brain ischemia and hemorrhage, respectively; 2) global bilateral ischemic changes (white changes) caused by the accompanying increase in intracranial pressure featuring brain ischemia followed by reperfusion and hyperemia; 3) five subcortical hematomas and two large bilateral subarachnoid hemorrhages with small subcortical hematomas, as confirmed on autopsy. The imaging detection limit was ~0.07 cc (5 mm in diameter) for saline and ~0.04 cc (4 mm in diameter) for blood at the estimated depth of 1 -1.5 cm. Our 1-min movie clip demonstrates the capability of optical imaging in detecting brain ischemia, hemorrhage, and reperfusion non-invasively and real-time, with high temporal and good spatial resolution. Fig 1. No caption Full figure and legend (64K)