Optically derived metabolic and hemodynamic parameters predict hippocampal neurogenesis in the BTBR mouse model of autism

In this study, we made use of dual‐wavelength laser speckle imaging (DW‐LSI) to assess cerebral blood flow (CBF) in the BTBR‐genetic mouse model of autism spectrum disorder, as well as control (C57Bl/6J) mice. Since the deficits in social behavior demonstrated by BTBR mice are attributed to changes in neural tissue structure and function, we postulated that these changes can be detected optically using DW‐LSI. BTBR mice demonstrated reductions in both CBF and cerebral oxygen metabolism (CMRO₂), as suggested by studies using conventional neuroimaging technologies to reflect impaired neuronal activation and cognitive function. To validate the monitoring of CBF by DW‐LSI, measurements with laser Doppler flowmetry (LDF) were also performed which confirmed the lowered CBF in the autistic‐like group. Furthermore, we found in vivo cortical CBF measurements to predict the rate of hippocampal neurogenesis, measured ex vivo by the number of neurons expressing doublecortin or the cellular proliferation marker Ki‐67 in the dentate gyrus, with a strong positive correlation between CBF and neurogenesis markers (Pearson, r = 0.78; 0.9, respectively). These novel findings identifying cortical CBF as a predictive parameter of hippocampal neurogenesis highlight the power and flexibility of the DW‐LSI and LDF setups for studying neurogenesis trends under normal and pathological conditions.      

Effects of head-down tilt on cerebral blood flow in humans ans rabbits

Changes in cerebral hemodynamics, during and after head down tilt (HDT), were examined by means of transcranial Doppler technique (TCD) and near infrared spectroscopy (NIRS) in humans, and laser Doppler flowmetry (LDF) in rabbits. Mean cerebral blood flow (CBF) velocity measured by TCD increased during the first 6 h of HDT compared with the pre-HDT value. NIRS experiments demonstrated that brain oxygenation and hemoglobin concentration increased with postural change from upright to supine. These results suggest that exposure to HDT increases CBF during the early phase of HDT in humans. In rabbits anesthetized with alpha chloralose, on the other hand, 45 degrees HDT did not change CBF significantly in the parietal cortex during 1 h after the onset of HDT. The discrepancy may be explained by the difference in species, tilt angle, or the brain region where CBF has been measured.     

α-Phenyl N-Tert-Butyl Nitrone (PBN) Increases the Cortical Cerebral Blood Flow by Inhibiting the Breakdown of Nitric Oxide in Anesthetized Rats

The effects of intravenous administration of a-phenyl N-tert-butyl nitrone (PBN) on cortical cerebral blood flow (CBF) were examined in Wistar rats under pentobarbital anesthesia and artificial ventilation. The cortical CBF in parietal cortex was measured by laser Doppler flowmetry. Intravenous administrations of 2 mg/kg and 20 mg/kg of PBN dose-dependently produced significant increases in cortical CBF and decreases in systemic blood pressure (BP). To examine whether these increased responses in cortical CBF produced by PBN were associated with the vasodilatation system of nitric oxide (NO), the NO synthase inhibitor L-N^G-nitroarginine (L-NOArg), which is an analog of L-arginine, was used to inhibit the NO-related-vasodilatative system. Since the PBN-induced responses in the cortical CBF were much attenuated in L-NOArg-treated rats (30 mg/kg, iv.), it was inferred that NO-related vasodilatation was strongly associated with the PBN-induced increase in cortical CBF.     

Circadian periodicity of cerebral blood flow revealed by laser-Doppler flowmetry in awake rats: relation to blood pressure and activity

Cardiovascular parameters such as arterial blood pressure (ABP) and heart rate display pronounced circadian variation. The present study was performed to detect whether there is a circadian periodicity in the regulation of cerebral perfusion. Normotensive Sprague-Dawley rats (SDR, approximately 15 wk old) and hypertensive (mREN2)27 transgenic rats (TGR, approximately 12 wk old) were instrumented in the abdominal aorta with a blood pressure sensor coupled to a telemetry system for continuous recording of ABP, heart rate, and locomotor activity. After 5-12 days, a laser-Doppler flow (LDF) probe was attached to the skull by means of a guiding device to measure changes in brain cortical blood flow (CBF). After the animals recovered from anesthesia, measurements were taken for 3-4 days. The time series were analyzed with respect to the midline estimating statistic of rhythm (i.e., mean value of a periodic event after fit to a cosine function), amplitude, and acrophase (i.e., phase angle that corresponds to the peak of a given period) of the 24-h period. The LDF signal displayed a significant circadian rhythm, with the peak occurring at around midnight in SDR and TGR, despite inverse periodicity of ABP in TGR. This finding suggests independence of LDF periodicity from ABP regulation. Furthermore, the acrophase of the LDF was consistently found before the acrophase of the activity. From the present data, it is concluded that there is a circadian periodicity in the regulation of cerebral perfusion that is independent of circadian changes in ABP and probably is also independent of locomotor activity. The presence of a circadian periodicity in CBF may have implications for the occurrence of diurnal alterations in cerebrovascular events in humans.     

Cerebral Blood Flow Modulation by Basal Forebrain or Whisker Stimulation Can Occur Independently of Large Cytosolic Ca2+ Signaling in Astrocytes

We report that a brief electrical stimulation of the nucleus basalis of Meynert (NBM), the primary source of cholinergic projection to the cerebral cortex, induces a biphasic cerebral cortical blood flow (CBF) response in the somatosensory cortex of C57BL/6J mice. This CBF response, measured by laser Doppler flowmetry, was attenuated by the muscarinic type acetylcholine receptor antagonist atropine, suggesting a possible involvement of astrocytes in this type of CBF modulation. However, we find that IP3R2 knockout mice, which lack cytosolic Ca2+ surges in astrocytes, show similar CBF changes. Moreover, whisker stimulation resulted in similar degrees of CBF increase in IP3R2 knockout mice and the background strain C57BL/6J. Our results show that neural activity-driven CBF modulation could occur without large cytosolic increases of Ca2+ in astrocytes.     

Treatment of Raynaud's phenomenon with ketanserin in patients with connective tissue disorders.

The serotonin receptor blocker ketanserin was given orally in a double blind crossover study to 10 patients with connective tissue disorders and Raynaud''s phenomenon. Eight of the 10 patients improved clinically on ketanserin and none on placebo. Digital blood flow was assessed with laser Doppler flowmetry (LDF), photoplethysmography, and skin temperature measurements. Laser Doppler flowmetry was the most useful method, showing a significant reduction in recovery time after a standard cold provocation. Although the resting flow was not significantly improved, digital ulcers healed in four out of five patients, providing evidence of increased nutritive flow. The results of this study suggest that orally administered ketanserin may be an effective and well tolerated treatment for Raynaud''s phenomenon associated with connective tissue disorders, especially scleroderma.     

Decreased cerebral blood flow and hemodynamic parameters during acute hyperglycemia in mice model observed by dual‐wavelength speckle imaging

In this study, we use dual‐wavelength optical imaging‐based laser speckle technique to assess cerebral blood flow and metabolic parameters in a mouse model of acute hyperglycemia (high blood glucose). The effect of acute glucose levels on physiological processes has been extensively described in multiple organ systems such as retina, kidney, and others. We postulated that hyperglycemia also alters brain function, which in turn can be monitored optically using dual‐wavelength laser speckle imaging (DW‐LSI) platform. DW‐LSI is a wide‐field, noncontact optical imaging modality that integrates the principles of laser flowmetry and oximetry to obtain macroscopic information such as hemoglobin concentration and blood flow. A total of eight mice (C57/BL6) were used, randomized into two groups of normoglycemia (control, n = 3) and hyperglycemia (n = 5). Hyperglycemia was induced by intraperitoneal injection of a commonly used anesthetic drug combining ketamine and xylazine (KX combo). We found that this KX combo increases blood glucose (BG) levels from 150 to 350 mg/dL, approximately, when measured 18 minutes post‐administration. BG continues to increase throughout the test period, with BG reaching an average of 463 ± 20.34 mg/dL within 60 minutes. BG levels were measured every 10 minutes from tail blood using commercially available glucometer. Experimental results demonstrated reductions in cerebral blood flow (CBF) by 55%, tissue oxygen saturation (SO₂) by 15%, and cerebral metabolic rate of oxygen (CMRO₂) by 75% following acute hyperglycemia. The observed decrease in these parameters was consistent with results reported in the literature, measured by a variety of experimental techniques. Measurements with laser Doppler flowmetry (LDF) were also performed which confirmed a reduction in CBF following acute hyperglycemia. In summary, our findings indicate that acute hyperglycemia modified brain hemodynamic response and induced significant changes in blood flow and metabolism. As far as we are aware, the implementation of the DW‐LSI to monitor brain hemodynamic and metabolic response to acute hyperglycemia in intact mouse brain has not been previously reported.      

Vascular changes associated with spinal root avulsion injury

Abstract

This paper has investigated the hypothesis that spinal root avulsion (SRA) injury produces alterations in blood flow that contribute to avulsion injury induced pain-like behaviour in rodents. Photoplethysmography (PPG) is an established way of assessing blood flow in the central nervous system (CNS) and laser Doppler flowmetry (LDF) is the most widely used technique for measuring tissue perfusion. Using an established model of SRA injury that produces mechanical hypersensitivity, the PPG and LDF signals were recorded in animals 2 weeks post-injury and compared to naive recordings. PPG and LDF measurements were assessed on the ipsilateral and contralateral sides of the spinal cord rostral and caudal to the avulsion injury and at the level of the injury. Two weeks after injury, a time when vascular blood vessel endothelial markers are known to be decreased, no significant changes were seen in the spinal cord blood flow (SCBF) above, at, or below the injury site or when comparing the ipsilateral vs. contralateral side. Assessment of oxygenation levels again revealed no significant differences between naive and spinal root injured animals along the rostrocaudal axis (i.e., above, at, and below the site of injury or its equivalent on the contralateral side). From these experiments it is concluded that SRA does not significantly alter blood flow or tissue oxygen levels and so ischemia may play a less prominent role in avulsion injury induced pain.     

Continual measurement of intramedullary blood perfusion with laser Doppler flowmetry in intact and ostectomized tibiae of rabbits

Blood flow is important for the healing of bone fractures. Until now, however, there have been no publications on the daily, continual measurement of intramedullary blood perfusion using laser Doppler flowmetry (LDF) in the conscious animal. In this study, a model for the daily, continual measurement of intramedullary blood perfusion by LDF and the temperature near the cortex both in intact and ostectomized tibiae in the conscious rabbit is described. The probes for blood perfusion and temperature measurement were implanted permanently at three different localizations into the right tibia of 10 adult New Zealand White rabbits. The probes were held in place by a bilateral, single-plane external fixator. In five of these animals, a midshaft tibial ostectomy was created in order to simulate a fracture. Intramedullary blood perfusion and temperature were measured daily over 49 days. While in intact tibiae no significant (P > 0.05) differences were found in blood perfusion readings taken at various time points, for mean values or for blood perfusion over time, in ostectomized tibiae the differences were significant: various time points (P = 0.0056), mean values (P = 0.0034) and blood perfusion over time (P = 0.0337). Blood perfusion readings at the centre probe were elevated compared with those at the proximal and distal probes. Thus, a revascularization in the ostectomy gap during the fracture healing was proven by means of the LDF. No influence of the blood perfusion on the temperature in the ostectomy area could be determined during healing of the ostectomy. The described model seems suitable for the continual measurement of intramedullary blood perfusion both in intact and ostectomized tibiae in the conscious rabbit.     

Capillary blood perfusion during postischemic reperfusion in striated muscle.

Monitoring of nutritive blood flow in muscle is of particular importance to reconstructive surgeons, since ischemia/reperfusion in striated muscle is known to result in postischemic microvascular perfusion failure. Laser Doppler flowmetry has recently been introduced as an easy-to-use, noninvasive technique for continuous monitoring of microvascular tissue perfusion. Despite its popularity, there exists a great deal of controversy as to what actually generates the laser Doppler signal recorded from a given tissue. Intravital microscopy is a technique for direct visualization of the nutritional circulation in tissue. By using intravital microscopy, direct measurements of blood perfusion in individual segments of the nutritional microcirculation can be made. In 22 Syrian golden hamsters we performed laser Doppler flowmetry and intravital microscopy measurements in muscle tissue prior to and during reperfusion after 4 hours of tourniquet ischemia using the dorsal skinfold chamber model. Intravital microscopy (n = 10) revealed a heterogeneous capillary perfusion during the early reperfusion phase with a decrease (p less than 0.01) in functional capillary density to 49.4 +/- 17.0 percent of control. No recovery was observed after 24 hours of reperfusion. Laser Doppler flowmetry (n = 12) showed a parallel reduction of capillary red blood cell flux during the early perfusion phase to 43.9 +/- 22.6 percent of control values (p less than 0.01), and no recovery was observed after 24 hours of reperfusion. However, the laser Doppler flowmetry technique was not able to detect the capillary perfusion inhomogeneities shown by intravital microscopy. Postischemic reperfusion in striated muscle is characterized by a decrease in functional capillary density and a heterogeneous capillary perfusion. Laser Doppler flowmetry is a useful tool for monitoring microvascular tissue perfusion, although in striated muscle of the hamster it must be considered that accurate nutritional "capillary" flow readings can be grossly overestimated if larger vessels, such as arterioles and collecting venules, are contained in the measuring field of the laser Doppler probe.     

Measurement of airway wall blood flow in sheep by laser-Doppler flowmetry: interpretation and problems

We have used laser-Doppler flowmetry (LDF), a technique that detects movement of erythrocytes, to measure tracheal and bronchial wall blood flow in anesthetized open-chest sheep. LDF derives continuous measurements noninvasively, although fiber-optic bronchoscopy is necessary to introduce the LDF probe into the airways. The response of the LDF flow signals at four regions of the airway walls to varying bronchial arterial flow rates was examined in both live and dead sheep by cannulation and subsequent perfusion of the common bronchial artery at different flow rates by use of a roller pump. In the live sheep, variations in bronchial arterial blood flow resulted in variations in LDF signals in the principal bronchus and in lobar and segmental bronchi but not in the trachea. In the dead sheep, variations in bronchial arterial blood flow resulted in variations in LDF signals in all four regions. Within regions, the average response of the LDF signals to varying bronchial blood flow rates was approximately linear in both live and dead sheep, but considerable site-to-site variation in response was observed. In the live sheep, significant LDF signals were observed when the bronchial arterial flow was set to zero and when the bronchial artery was perfused with dextran solution, which would in theory be expected to produce no LDF signal. A small LDF signal was also detected under zero flow conditions in the dead sheep. These observations suggest that the LDF technique, in addition to detecting blood flow from the bronchial artery also detects background noise and/or collateral circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic evaluation of blood flow microcirculation by combined use of the laser Doppler flowmetry and high‐speed videocapillaroscopy methods

The dynamic light scattering methods are widely used in biomedical diagnostics involving evaluation of blood flow. However, there exist some difficulties in quantitative interpretation of backscattered light signals from the viewpoint of diagnostic information. This study considers the application of the high‐speed videocapillaroscopy (VCS) method that provides the direct measurement of the red blood cells (RBCs) velocity into a capillary. The VCS signal presents true oscillation nature of backscattered light caused by moving RBCs. Thus, the VCS signal can be assigned as a reference one with respect to more complicated signals like in laser Doppler flowmetry (LDF). An essential correlation between blood flow velocity oscillations in a separate human capillary and the integral perfusion estimate obtained by the LDF method has been found. The observation of blood flow by the VCS method during upper arm occlusion has shown emergence of the reverse blood flow effect in capillaries that corresponds to the biological zero signal in the LDF. The reverse blood flow effect has to be taken into account in interpretation of LDF signals.      

Laser Doppler flowmeter monitoring of free-tissue transfers: blood flow in normal and complicated cases

In this article, laser Doppler flowmeter (LDF) monitoring of blood flow in 94 free flaps is summarized. Seventy-six patients had uneventful postoperative courses, and 18 patients developed postoperative complications, with a salvage rate of 88 percent. Except for one case, the flowmeter identified developing complications before clear clinical indices appeared, and in two cases it was the only indication of vascular compromise of the flap. On the basis of the data obtained, the ranges of absolute flow values in different types of uncomplicated flaps are reported, along with their temporal pattern of flow. Decrease inflow pattern may be an early indicator of a developing perfusion disturbance. On the basis of LDF readings, the following classifications of free-flap blood flow are suggested. (1) If the flow is within or slightly above the established range, then normal diligence in observation is justified. If the flow is well above the normal range, artifacts that could falsely elevate readings should be investigated. (2) If the flow is somewhat below the established range, then a modest increase in observation is warranted (alert level 1). (3) If the relative flow falls to 50 percent of the initial flow of that flap and remains at that level for 30 minutes or longer, then more aggressive flap observation is indicated (alert level 2). (4) If the flow is below 0.4 LDF units for 30 minutes, then aggressive clinical observation should be performed (alert level 3, or "red alert") and exploration should be strongly considered.Falsely elevated measurements can be caused by vibration, motion of the probe or tissue, or location of the probe over a macroscopic blood vessel. False low readings are quite rare but can result from partial probe detachment from the flap or coagulum accumulating on the probe. Once artifacts are ruled out, LDF readings have a high level of credibility and, in the authors' experience, significantly improve salvage rates.     

Differing effects of vasopressin on regional cerebral blood flow of dogs following intracisternal vs. intra-arterial administration

We investigated the differential effect of the intracisternal and intraarterial administration of vasopressin on the regional cerebral blood flow (rCBF) in the parietal cortex of dogs. Regional CBF, velocity and blood volume were assayed by laser flowmetry. The intracisternal injection of 1 nmol vasopressin significantly increased the rCBF and velocity, without affecting blood volume. However, the intravertebral arterial injection of 1 nmol vasopressin significantly decreased the rCBF and velocity. This discrepancy can be explained by a difference in the affected vasculature; large blood vessels in the subarachnoid space vs. whole cerebral vascular system. The intracisternal and intraarterial injection of the nitric oxide inhibitor N^G-monomethyl-L-arginine reduced the rCBF from the base line, and significantly suppressed the rCBF elevation induced by vasopressin. The effect of vasopressin may be considered as the summation of the increased flow from the dilated large vessels via the release of nitric oxide from the endothelium, and of the decreased flow from the contracted small vessels.     

The use of sensory-sympathetic coupling indices for diagnosing of sympathetically maintained pain with laser Doppler flowmetry

For the first time the objective diagnosis of sympathetically maintained pain was created with laser Doppler flowmetry (LDF), directed specially to discovery of skin sensory-sympathetic coupling at 49 patients with posttraumatic complex regional pain syndrome. Sensory-sympathetic coupling was diagnosed as combination of sympathetic vasomotor activity with the existence of sensory peptidergic blood flow oscillations in frequency range of 0.047-0.069 Hz in LDF wavelet-spectrum. The results of LDF diagnosis were compared with clinical evaluation of sympathetically maintained pain carried out after desympathization surgery (thoracoscopic clipping above and below the Th3 ganglion of sympathetic chain at 33 patients and perivascular sympathectomy at the level of brachial artery and veins at 16 patients). Sensitivity of preoperative LDF-diagnosis was 90.2%, specificity--87.5%, positive predictive value--97.3%, negative predictive value--63.6%, diagnostic effectiveness--89.8%.     

Use of sensory-sympathetic coupling indices for diagnosing sympathetically maintained pain with laser Doppler flowmetry

The first objective diagnosis of sympathetically maintained forms of pain employing laser Doppler flowmetry (LDF) and specifically targeted at detecting sensory-sympathetic coupling in the skin has been developed and tested in 49 patients with the posttraumatic complex regional pain syndrome. Sensory-sympathetic coupling was diagnosed as a combination of sympathetic vasomotor activity and the presence of sensory peptidergic blood flow oscillations in a frequency band of 0.047–0.069 Hz in the LDF wavelet spectrum. The results of the LDF diagnosis were compared with the clinical evaluation of sympathetically maintained pain carried out after desympathization surgery (thoracoscopic clipping above and below the Th3 ganglion of the sympathetic chain in 33 patients and perivascular sympathectomy at the level of the brachial vascular bundle in 16 patients). The sensitivity of preoperative LDF diagnosis was 90.2%; the specificity was 87.5%; the positive predictive value was 97.3%; the negative predictive value was 63.6%; and the diagnostic efficiency was 89.8%.     

Short duration hyperbaric oxygen treatment effects blood flow in rats: pilot observations

Hyperbaric oxygen (HBO) treatment has been found to improve healing in living tissues, especially those poor in oxygen. The effects of HBO have also been tested in rat experiments. However, oxygen partial pressure in rat's arterial blood is normally about twice that in humans. Disregarding this, a human HBO protocol has been applied in previous rat experiments with HBO. Laser Doppler flowmetry (LDF) is a non-invasive means for measuring blood flow. Using LDF, we measured the blood perfusion rate in rats receiving HBO, according to a modified protocol, in a region of healing soft tissue with bone defect. The results indicate that, in rats, shorter HBO treatment with high O2 pressure can significantly improve the blood flow of healing tissues. In this study, an elevated blood perfusion rate was still evident 2 weeks after the ending of HBO therapy, which indicates improved revascularization in the wound area. A short HBO protocol would save time and effort in future HBO experiments on rats.     

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 μm in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 μm below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.     

Adaptive wavelet analysis of oscillations in the human peripheral blood flow

The main principles are outlined for spectral timing analysis of the peripheral blood flow oscillations obtained by laser Doppler flowmetry. The oscillations can be studied in a wide frequency range both in stationary and nonstationary conditions during functional tests. The potential of the method has been demonstrated in experiments with the reaction of the microvascular bed to transcutaneous iontophoretic introduction of acetylcholine chloride. The major advantage of the method over conventional wavelet analysis is a significant increase in the “effective” length of the signal analyzed, which allows correct analysis of low-frequency components in much shorter LDF recordings than those commonly used.     

Early circulatory and metabolic events in island skin flaps of the pig

Circulatory and metabolic skin-flap events were studied prior to and up to 6 hours after elevation of buttock island flaps in pigs. During the elevation, significant reductions in superficial skin blood flow, measured by laser Doppler flowmetry (LDF) and dermal flap temperature, were seen. Significant correlations were found between blood flow and temperature. Total flap blood flow, measured as venous outflow, also showed an initial transient decrease, but 2 hours after flap construction, venous outflow had returned to preoperative values. A significant increase in lactate release, together with increased oxygen consumption and glucose uptake, was seen 4 hours after the surgical intervention. Hypoxanthine release, indicating ischemia, was seen only during the first hour after flap elevation. Noradrenaline outflow was noted after 4 and 6 hours, but there was no parallel reduction in flap blood flow. A great deal of the flow reduction in acutely elevated island flaps may thus be due to primary hypothermia rather than to the degenerative release of noradrenaline, which seems to have no early effect on skin flap blood flow. On the other hand, the noradrenaline release may be linked to an increased metabolic activity in the skin flaps.