Window Preparations Research Articles

In vivo Two-Photon Imaging of Anesthesia-Specific Alterations in Microglial Surveillance and Photodamage-Directed Motility in Mouse Cortex

Two-photon imaging of fluorescently labeled microglia in vivo provides a direct approach to measure motility of microglial processes as a readout of microglial function that is crucial in the context of neurodegenerative diseases, as well as to understand the neuroinflammatory response to implanted substrates and brain-computer interfaces. In this longitudinal study, we quantified surveilling and photodamage-directed microglial processes motility in both acute and chronic cranial window preparations and compared the motility under isoflurane and ketamine anesthesia to an awake condition in the same animal. The isoflurane anesthesia increased the length of surveilling microglial processes in both acute and chronic preparations, while ketamine increased the number of microglial branches in acute preparation only. In chronic (but not acute) preparation, the extension of microglial processes toward the laser-ablated microglial cell was faster under isoflurane (but not ketamine) anesthesia than in awake mice, indicating distinct effects of anesthetics and of preparation type. These data reveal potentiating effects of isoflurane on microglial response to damage, and provide a framework for comparison and optimal selection of experimental conditions for quantitative analysis of microglial function using two-photon microscopy in vivo.

Abstract 101: ApoE4 Disrupts Cerebrovascular Microcirculation and Undermines White Matter Integrity and Cognitive Function

Background: Small vessel disease leading to subcortical white matter lesions (WML) is the major cause of vascular cognitive impairment. The apolipoprotein E4 (ApoE4) allele, a well-known risk factor for Alzheimer’s disease, is also a risk factor for WML, but the mechanisms of the effect remain unclear. We set out to determine whether the ApoE4 genotype disrupts the cerebral microcirculation and promotes WML and cognitive impairment. Methods: We studied male ApoE4 targeted replacement mice (ApoE4-TR; age 3-4 months). ApoE3 (ApoE3-TR) were used as controls. Cerebral blood flow (CBF) was assessed by ASL-MRI; the CBF increase induced by whisker stimulation (WS) or the endothelium-dependent agent acetylcholine (ACh) was assessed in cranial window preparations by laser-Doppler flowmetry. The corpus callosum (CC) microcirculation was examined by three-photon microscopy (3PM). Bilateral carotid artery stenosis (BCAS) was induced with 0.18 mm microcoils. WML and cognition were tested 4 weeks after BCAS. Results: In ApoE4-TR mice resting cortical CBF was attenuated compared to ApoE3-TR (-13%), as well the increase in CBF produced by WS (-47±3%) or ACh (-38±3%; p<0.05; n=5/group). In the CC, red blood cell (RBC) speed was slower in ApoE4-TR (1.6±0.1) than in ApoE3 mice (1.9±0.1 mm/s; p<0.05; n=5/group). Following 4 weeks of BCAS, cortical CBF was reduced more in ApoE4-TR (-35±4%) than in ApoE3-TR mice (-23±2%; p<0.05; n=8/group). Similarly, in the CC RBC speed was reduced more in ApoE4-TR (-33±3%) than in ApoE3-TR mice (-23±3%; p<0.05; n=5/group). In ApoE4-TR mice BCAS resulted in more marked CC WML (SMI312/MBP ratio, 48%) and cognitive dysfunction (novel object recognition: 18%; Y-maze arm alternation: 14%) than in ApoE3-TR mice (p<0.05; n=8/group). Conclusions: The findings unveil a previously unrecognized ApoE4-dependent failure of critical neurovascular regulatory mechanisms, sufficient to promote white matter damage in the setting of cerebral hypoperfusion. Such alterations may be responsible for the increased susceptibility to hypoxic-ischemic lesions in the subcortical white matter of individuals carrying the ApoE4 allele.

Oral Metoprolol Attenuates Dilation of Leptomeningeal Collateral Arteries after Middle Cerebral Artery Occlusion and Increases Infarct Volume in Sprague Dawley Rats

BackgroundBeta‐adrenergic receptor antagonists, or beta blockers (BBs), have long been a mainstay in the treatment of hypertension, heart failure, and angina. In 2014, the Eighth Joint National Committee removed BBs from the list of recommended first‐line hypertension therapies, citing an increased incidence of stroke among patients treated with BBs compared to other antihypertensive medications. However, in 2015, more than 72 million prescriptions of this drug class were filled in the United States. The mechanism behind worse stroke outcomes in BB‐treated patients is unknown. Leptomeningeal collateral arteries (LCAs) connect adjacent arterial trees of the cerebral circulation, and after ischemic stroke, provide an alternative pathway for blood flow to the affected cortical tissue. We hypothesize that beta‐adrenergic receptor‐mediated dilation of LCAs is an important part of this protective mechanism during ischemic stroke and that BBs attenuate this dilation, leading to worse stroke outcomes.Methods and ResultsLCAs dilated in a concentration‐dependent manner to topical application of the beta‐adrenergic receptor agonist isoproterenol in cranial window preparations in Sprague Dawley (SD) rats. Isolated, pressurized LCAs dilated in response to isoproterenol ex vivo, and this dilation was attenuated by the beta1‐selective blocker CGP20712 (30 nM). LCAs acutely dilated after middle cerebral artery occlusion (MCAO) in SD rats. The dilation of LCAs after MCAO was reduced in rats suffused with topical CGP20712 (100 nM) or gavaged with oral metoprolol (30 mg/kg), the most frequently prescribed BB. Similarly, blood flow through LCAs post‐MCAO was reduced in rats gavaged with metoprolol compared to control rats, as visualized by laser speckle contrast imaging. Finally, infarct volume, as measured by MRI one day post‐MCAO surgery, was larger in rats gavaged with metoprolol compared to control. Interestingly, the heart rate and systemic blood pressure measured by biotelemetry for 6 hours following MCAO surgery were not different in rats gavaged with metoprolol and water.ConclusionsOur study suggests that BBs may promote worse stroke outcomes by disrupting beta‐adrenergic receptor‐mediated dilation of LCAs.Support or Funding InformationSupported by NIH R01 HL097107, American Heart Association 17GRNT33670970, and Institutional Hornick and Fund‐to‐Cure‐Stroke grantsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spatio-temporal dynamics of cerebral capillary segments with stalling red blood cells

Optical coherence tomography (OCT) allows label-free imaging of red blood cell (RBC) flux within capillaries with high spatio-temporal resolution. In this study, we utilized time-series OCT-angiography to demonstrate interruptions in capillary RBC flux in mouse brain in vivo. We noticed ∼7.5% of ∼200 capillaries had at least one stall in awake mice with chronic windows during a 9-min recording. At any instant, ∼0.45% of capillaries were stalled. Average stall duration was ∼15 s but could last over 1 min. Stalls were more frequent and longer lasting in acute window preparations. Further, isoflurane anesthesia in chronic preparations caused an increase in the number of stalls. In repeated imaging, the same segments had a tendency to stall again over a period of one month. In awake animals, functional stimulation decreased the observance of stalling events. Stalling segments were located distally, away from the first couple of arteriolar-side capillary branches and their average RBC and plasma velocities were lower than nonstalling capillaries within the same region. This first systematic analysis of capillary RBC stalls in the brain, enabled by rapid and continuous volumetric imaging of capillaries with OCT-angiography, will lead to future investigations of the potential role of stalling events in cerebral pathologies.

Characterization of the BAC Id3-enhanced green fluorescent protein transgenic mouse line for in vivo imaging of astrocytes.

Astrocytes are highly ramified glial cells with critical roles in brain physiology and pathology. Recently, breakthroughs in imaging technology have expanded our understanding of astrocyte function in vivo. The in vivo study of astrocytic dynamics, however, is limited by the tools available to label astrocytes and their processes. Here, we characterize the bacterial artificial chromosome transgenic Id3-EGFP knock-in mouse to establish its usefulness for in vivo imaging of astrocyte processes. Using fixed brain sections, we observed enhanced green fluorescent protein expression in astrocytes and blood vessel walls throughout the brain, although the extent and cell type specificity of expression depended on the brain area and developmental age. Using in vivo two-photon imaging, we visualized astrocytes in cortical layers 1-3 in both thin skull and window preparations. In adult animals, astrocytic cell bodies and fine processes could be followed over many hours. Our results suggest that Id3 mice could be used for in vivo imaging of astrocytes and blood vessels in development and adulthood.

Evaluation of cranial window types forin vivotwo-photon imaging of brain microstructures

In vivo two-photon laser scanning microscopy (2PLSM) of neurons and glia in the mouse neocortex can be achieved using two distinct surgical methods: the thin-skull and open-skull window techniques. The open-skull window technique is more invasive, but provides more flexibility in imaging periods and frequencies. Critical evaluations of the two surgical methods are required, particularly their ability to image submicron structures such as dendritic filopodia and spines. Here, we used multiple approaches to evaluate the optical resolution of 2PLSM through two types of cranial windows. Two-photon point spread function (PSF) on the surface of the neocortex in vivo was studied by imaging the fluorescent microspheres attached to the pial surface. Comparable PSF widths indicated that the optical resolution of 2PLSM on the brain surface was similar between the two types of cranial window techniques. Next, we evaluated the effects of laser beam propagation through the thinned bone by imaging the underlying gel samples containing fluorescent microspheres as phantoms mimicking the brain tissue. We observed deterioration of PSF with increase in the imaging depths through the thinned bone. Reduction in image resolution through the thin-skull window in the deep cortical layer was confirmed by repetitive 2PLSM of the same GFP-positive dendritic spines with two techniques. In summary, the resolution of 2PLSM is comparable with those of two cranial window preparations in the superficial layer of the neocortex, but the quality of the image through the thin-skull windows degrades at points deeper than 50 µm.

Brain surface temperature under a craniotomy

Many neuroscientists access surface brain structures via a small cranial window, opened in the bone above the brain region of interest. Unfortunately this methodology has the potential to perturb the structure and function of the underlying brain tissue. One potential perturbation is heat loss from the brain surface, which may result in local dysregulation of brain temperature. Here, we demonstrate that heat loss is a significant problem in a cranial window preparation in common use for electrical recording and imaging studies in mice. In the absence of corrective measures, the exposed surface of the neocortex was at ∼28°C, ∼10°C below core body temperature, and a standing temperature gradient existed, with tissue below the core temperature even several millimeters into the brain. Cooling affected cellular and network function in neocortex and resulted principally from increased heat loss due to convection and radiation through the skull and cranial window. We demonstrate that constant perfusion of solution, warmed to 37°C, over the brain surface readily corrects the brain temperature, resulting in a stable temperature of 36-38°C at all depths. Our results indicate that temperature dysregulation may be common in cranial window preparations that are in widespread use in neuroscience, underlining the need to take measures to maintain the brain temperature in many physiology experiments.

Effect of static and cyclic loading on ceramic laminate veneers adhered to teeth with and without aged composite restorations.

Existing composite restorations on teeth are often remade prior to the cementation of fixed dental prostheses. The aim of this study was to evaluate the effect of static and cyclic loading on ceramic laminate veneers adhered to aged resin composite restorations. Eighty sound maxillary incisors were collected and randomly divided into four groups: group 1: control group, no restorations; group 2: two Class III restorations; group 3: two Class IV restorations; group 4: complete composite substrate. Standard composite restorations were made using a microhybrid resin composite (Anterior Shine). Restored teeth were subjected to thermocycling (6000 cycles). Window preparations were made on the labial surface of the teeth for ceramic laminate fabrication (Empress II). Teeth were conditioned using an etch-and-rinse system. Existing composite restorations representing the aged composites were silica coated (CoJet) and silanized (ESPE-Sil). Ceramic laminates were cemented using a bis-GMA-based cement (Variolink Veneer). The specimens were randomly divided into two groups and were subjected to either static (groups 1a, 2a, 3a, 4a) or cyclic loading (groups 1b, 2b, 3b, 4b). Failure type and location after loading were classified. Data were analyzed using one-way ANOVA and Tukey's test. Significantly higher fracture strength was obtained in group 4 (330 ± 81 N) compared to the controls in group 1 (179 ± 120 N) (one-way ANOVA, p < 0.05). Group 1b survived a lower mean number of cyclic loads (672,820 cycles) than teeth of groups 2b to 4b (846x103 to 873x103 cycles). Failure type evaluation after the fracture test showed predominantly adhesive failures between dentin and cement, but after cyclic loading, more cohesive fractures in the ceramic were seen. Ceramic laminate veneers bonded to conditioned aged composite restorations provided favorable results. Surface conditioning of existing restorations may eliminate the necessity of removing aged composite restorations.

In Vivo Imaging of Tumors

Light microscopy of tumors, as for other thick, scattering tissues such as the brain or the developing embryo, is limited by light penetration and optical access. because of these problems, epifluorescence and confocal microscopy are typically limited to the outer 50-100 microm of the accessible tumor tissue. Most mouse tumors must be exteriorized for examination under the light microscope, a procedure that limits the duration and repeatability of imaging. this protocol describes the generation of chronic window preparations in the mouse. These preparations allow an implanted tumor to grow for several weeks in an optically accessible location in vivo, making it possible to examine the living tumor with high-resolution light microscopy in a repetitive manner. Two chronic window preparations are described: (1) the dorsal skinfold chamber, which allows in vivo imaging of tumors growing in the subcutaneous space, and (2) the cranial window, which allows in vivo imaging of tumors growing on the brain surface.

Quantitative cerebral blood flow with Optical Coherence Tomography

Absolute measurements of cerebral blood flow (CBF) are an important endpoint in studies of cerebral pathophysiology. Currently no accepted method exists for in vivo longitudinal monitoring of CBF with high resolution in rats and mice. Using three-dimensional Doppler Optical Coherence Tomography and cranial window preparations, we present methods and algorithms for regional CBF measurements in the rat cortex. Towards this end, we develop and validate a quantitative statistical model to describe the effect of static tissue on velocity sensitivity. This model is used to design scanning protocols and algorithms for sensitive 3D flow measurements and angiography of the cortex. We also introduce a method of absolute flow calculation that does not require explicit knowledge of vessel angles. We show that OCT estimates of absolute CBF values in rats agree with prior measures by autoradiography, suggesting that Doppler OCT can perform absolute flow measurements in animal models.

Increased Extracellular K + Concentration Reduces the Efficacy of N -methyl- d -aspartate Receptor Antagonists to Block Spreading Depression-Like Depolarizations and Spreading Ischemia

Spreading depression (SD)-like depolarizations may augment neuronal damage in neurovascular disorders such as stroke and traumatic brain injury. Spreading ischemia (SI), a particularly malignant variant of SD-like depolarization, is characterized by inverse coupling between the spreading depolarization wave and cerebral blood flow. SI has been implicated in particular in the pathophysiology of subarachnoid hemorrhage. Under physiological conditions, SD is blocked by N-methyl-D-aspartate receptor (NMDAR) antagonists. However, because both SD-like depolarizations and SI occur in presence of an increased extracellular K+ concentration ([K+]o), we tested whether this increase in baseline [K+]o would reduce the efficacy of NMDAR antagonists. Cranial window preparations, laser Doppler flowmetry, and K+-sensitive/reference microelectrodes were used to record SD, SD-like depolarizations, and SI in rats in vivo; microelectrodes and intrinsic optical signal measurements were used to record SD and SD-like depolarizations in human and rat brain slices. In vivo, the noncompetitive NMDAR antagonist dizocilpine (MK-801) blocked SD propagation under physiological conditions, but did not block SD-like depolarizations or SI under high baseline [K+]o. Similar results were found in human and rat neocortical slices with both MK-801 and the competitive NMDAR antagonist D-2-amino-5-phosphonovaleric acid. Our data suggest that elevated baseline [K+]o reduces the efficacy of NMDAR antagonists on SD-like depolarizations and SI. In conditions of moderate energy depletion, as in the ischemic penumbra, or after subarachnoid hemorrhage, NMDAR inhibition may not be sufficient to block these depolarizations.

Chronic nicotine alters NO signaling of Ca(2+) channels in cerebral arterioles.

Smoking is a major health hazard with proven deleterious effects on the cerebral circulation, including a decrease in cerebral blood flow and a high risk for stroke. To elucidate cellular mechanisms for the vasoconstrictive and pathological effects of nicotine, we used a nystatin-perforated patch-clamp technique to study Ca(2+) channels and Ca(2+)-activated K(+) (BK) channels in smooth muscle cells isolated from cerebral lenticulostriate arterioles of rats chronically exposed to nicotine (4.5 mg/kg per day of nicotine free base, 15 to 22 days via osmotic minipump). Two major effects were observed in cells from nicotine-treated animals compared with controls. First, Ca(2+) channels were upregulated (0.48+/-0.03 pS/pF [20 cells] versus 0.35+/-0.01 pS/pF [31 cells], P:<0.005) and BK channels were downregulated (12+/-3 pA/pF [14 cells] versus 34+/-7 pA/pF [14 cells], P:<0.05), mimicking the effect of an apparent decrease in bioavailability of endogenous NO. Second, normal downregulation of Ca(2+) channels by exogenous NO (sodium nitroprusside [SNP], 100 nmol/L) and cGMP (8-bromo-cGMP, 0.1 mmol/L) was absent, whereas normal upregulation of BK channels by these agents was preserved, suggesting block of NO signaling downstream of cGMP-dependent protein kinase. In pial window preparations, chronic nicotine blunted NO-induced vasodilation of pial vessels and the increase in cortical blood flow measured by laser-Doppler flowmetry, demonstrating the importance of Ca(2+) channel downregulation in NO-induced vasorelaxation. These findings elucidate a new pathophysiological mechanism involving altered Ca(2+) homeostasis in cerebral arterioles that may predispose to stroke.

Investigating pulsed dye laser-blood vessel interaction with color Doppler optical coherence tomography

A non-invasive method of imaging laser irradiated blood vessels has been achieved using Color Doppler Optical Coherence Tomography (CDOCT). This method may increase understanding of the mechanisms behind treatment of vascular disorders. The CDOCT system used a 1280 nm center wavelength superluminescent diode. A 585 nm, 360 aes pulsed dye laser was used to irradiate hamster dorsal skin flap window preparations. Irradiation sites were imaged with CDOCT prior to, immediately after, and 24 hours after laser irradiation. The processed CDOCT signal provided an estimate of the blood flow velocity. An increase in the blood vessel backscattered signal was observed as blood or vessel walls were coagulated. A decrease in damaged blood vessel reflectivity occurred after twenty four hours.

Traumatic brain injury does not alter cerebral artery contractility

Previous studies have shown that traumatic brain injury (TBI) significantly reduces cerebral blood flow determined in vivo and reduces vascular reactivity in the pial circulation measured with cranial window preparations. We have now tested the hypothesis that TBI induces these changes by impairing intrinsic contractile activity of cerebral arteries. Anesthetized rats underwent moderate (2.2 atm) and severe (3.0 atm) midline fluid percussion TBI or sham injury following which posterior cerebral or middle cerebral arteries were isolated and isometric force generation was measured. Moderate (n = 5) and severe (n = 3) trauma had no effect on the magnitude of serotonin- or K+-induced force generation or sensitivity to serotonin in arteries isolated within 10 min of TBI. Functional disruption of the endothelium of posterior cerebral arteries isolated 10 min after moderate trauma or sham injury caused a reduction in the active tension response to serotonin that was similar in both groups. Blockade of cyclooxygenase with 5 microM indomethacin had no effect on serotonin-induced force generated by vessels with moderate trauma or in sham-treated rats. Acetylcholine induced an endothelium-dependent relaxation of posterior and middle cerebral arteries; the magnitude of the response was unaffected by moderate TBI. To determine whether prolonged in situ exposure of vessels to the traumatized cerebral milieu could reveal an alteration in intrinsic contractility, posterior cerebral arteries were isolated 30 min after TBI; again, no differences in the tension or relaxation responses were observed. It is concluded that midline fluid percussion TBI did not affect contraction or relaxation of proximal middle or posterior cerebral arteries in rats.

Regional cerebral blood flow response to vibrissal stimulation in mice lacking type I NOS gene expression.

The role of nitric oxide (NO) in cerebral blood flow-metabolism coupling was assessed in SV-129 wild-type (WT) and neuronal (type I) NO synthase (NOS) knockout mice (Kn). Regional cerebral blood flow (rCBF; laser-Doppler flowmetry) was measured over the contralateral cortical barrel field during unilateral mechanical vibrissal deflection (2-3 Hz, 60 s) under urethan anesthesia. The rCBF response was similar in WT and Kn and did not differ when recorded over the intact skull or closed cranial window preparations. Whisker stimulation increased rCBF by 41 +/- 8% (maximum) and 27 +/- 6% (mean) in WT (n = 6) and 41 +/- 7% (maximum) and 26 +/- 6% (mean) in Kn (n = 6) when recorded through a closed cranial window. After superfusion with topical N omega-nitro-L-arginine (L-NNA; 1 mM), the rCBF response was inhibited by approximately 45% in WT mice (P < 0.05), whereas there was no inhibition in Kn. Endothelium-dependent relaxation, assessed by pial vessel dilation in response to topical acetylcholine (100 microM) and inhibition by L-NNA (1 mM), was the same in both groups. Our results suggest that 1) endothelial NO production does not mediate the rCBF coupling to neuronal activity in Kn, 2) the inhibitory effect of L-NNA on the rCBF response to whisker stimulation in WT is a consequence of type I (neuronal) NOS inhibition, and 3) NO-independent mechanisms couple rCBF and metabolism during whisker stimulation in mice lacking expression of neuronal NOS.

Nitric oxide synthase inhibition irreversibly decreases perfusion in the R3230Ac rat mammary adenocarcinoma

We examined the microvascular effects of competitive nitric oxide synthase (NOS) inhibition with NG-monomethyl-L-arginine (MeArg), followed by L-arginine, on R3230Ac mammary adenocarcinoma perfusion. In window preparations containing tumours, superfusion of 50 microM MeArg reduced diameters of central tumour venules by 13%, of peripheral tumour venules by 17% and of normal venules near tumours by 16% from baseline. MeArg reduced red blood cell (RBC) velocity in central tumour venules by 25%, and increased intermittent flow and stasis frequency by 20% in central tumour venules. Subsequent superfusion of 200 microM L-arginine did not restore diameters or RBC velocity of any tumour preparation venules, and decreased length density in both central tumour venules and peripheral tumour venules. In contrast, MeArg reduced control preparation venule diameter by 30% and RBC velocity by 66%, but did not decrease length density or increase intermittent flow or stasis frequency. Unlike tumour preparation venules, L-arginine restored control venule diameters and velocities. NOS inhibition reduces both tumour and control venule perfusion, but the effect is blunted in the vicinity of tumours, possibly because of increased NOS levels. Perfusion can be subsequently restored in control, but not tumour, venules with L-arginine. Tumour NOS inhibition, followed by normal tissue rescue with L-arginine, may provide a novel means to achieve the goal of selective tumour hypoxia.

Disruption of the blood-brain barrier in open and closed cranial window preparations in rats.

The goal of this study was to determine whether the severity of disruption of the blood-brain barrier during acute hypertension is similar in open and closed cranial window preparations. Intravital fluorescent microscopy and fluorescein-labeled albumin were used to evaluate disruption of the blood-brain barrier under control conditions and during acute arterial hypertension in 10 rats equipped with an open cranial window and in six rats equipped with a closed cranial window. Permeability of the blood-brain barrier was quantified by calculating the clearance of fluorescein-labeled albumin and by counting the number of microvascular leaky sites under control conditions and during acute hypertension. Pressure in cerebral venules and intracranial pressure were measured in rats equipped with an open cranial window and a closed cranial window, respectively, under control conditions and during acute hypertension. In rats equipped with an open cranial window, arterial pressure increased from 118 +/- 6 to 189 +/- 3 mm Hg (mean +/- SEM) and pial venous pressure increased from 7 +/- 1 to 22 +/- 3 mm Hg during acute hypertension induced with 30 micrograms/kg/min phenylephrine for 5 minutes. In addition, the clearance of fluorescent albumin increased from 0.11 +/- 0.03 to 1.2 +/- 0.4 ml/sec x 10(-6) and the number of microvascular leaky sites increased from 0 to 25 +/- 1 during phenylephrine infusion. In rats equipped with a closed cranial window, arterial pressure increased from 122 +/- 5 to 187 +/- 7 mm Hg and intracranial pressure increased from 3 +/- 1 to 12 +/- 1 mm Hg during the intravenous infusion of 30 micrograms/kg/min phenylephrine for 5 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of burn injury on vasoactivity in hamster peripheral microcirculation

The effects of mild thermal shock on the vasoactivity of microvessels were studied in the hamster skin flap window preparations. Diameter fluctuations in arterioles and venules, varying in size from 10 to 50 μm and at different branching order sites, were measured prior and subsequent to a local surface skin burn. The experimental vasomotion data were characterized in terms of mean, standard deviation, skewness, and kurtosis, and by the Prony spectral line estimator (PSLE), fast Fourier transform (FFT), and auto regression (AR) methods of spectral analysis. Following a mild burn the A1 (37–50μm), A2 (30–50 μm), and A3 (25–41 μm) arterioles relaxed to a larger diameter by an average of 58, 20, and 13%, respectively. Dispersion statistics postburn showed a skewness close to normal while the kurtosis became more negative, indicating that the transient diameter curves were flatter. Both FFT and PSLE analyses indicate less energy in the signals postburn accompanied by a shift toward lower frequencies with decreased amplitude. In addition, there was a loss of certain frequencies from the spectrum and an increase in the interval for rhythmic activity. FFT analysis gave an idea of the trend while PSLE analysis was found to be highly unstable and dependent on the window size. AR results indicate that the process is stable and definite trends exist in the data, however, the data are not purely periodic.

Ultrastructure of skin window cells in malignant lymphoma.

The ultrastructure of cells in skin window preparations was analysed in 38 patients with malignant lymphoma and in 22 normal subjects. In the series as a whole, there were significant morphological changes in the macrophages between day 1 and day 2 consistent with cell maturation during the inflammatory response. At both intervals, lymphoma patients showed strikingly larger cell and nuclear profile areas as compared with normal subjects; there was a greater number of nucleoli per profile and proportionately more sections showed a Golgi complex. At day 1, a greater percentage of the patients' macrophages showed large numbers of phagocytic inclusions, but at day 2, significantly fewer profiles demonstrated this feature. These morphological findings could be a reflection of the known mononuclear phagocyte dysfunction in malignant lymphoma.

Examination of skin window preparations by transmission electron microscopy.

Examination of skin window preparations by transmission electron microscopy.