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The Effect of the Cold Pressor Test on a Visually Evoked Cerebral Blood Flow Velocity Response

      Abstract

      We investigated the hypothesis that during tonic pain stimulus, neurovascular coupling (NVC) decreases, measuring visually evoked cerebral blood flow velocity response (VEFR) during cold pressor test (CPT) in healthy human subjects as a test. VEFR was calculated as a relative increase in blood flow velocity in the posterior cerebral artery from average values during the last 5 s of the stimulus-OFF period to average values during the last 10 s of the stimulus-ON period. Three consecutive experimental phases were compared: basal, CPT and recovery. During CPT, end-diastolic and mean VEFR increased from 20.2 to 23.6% (p < 0.05) and from 17.5 to 20.0% (p < 0.05), respectively. In recovery phase, end-diastolic and mean VEFR decreased to 17.7% and 15.5%, respectively. Both values were statistically significantly different from CPT phase (p < 0.05). Compared with the basal phase, only end-diastolic VEFR was statistically significantly different in the recovery phase (p < 0.05). Our results are consistent with the assumption that there is a change in the activity of NVC during CPT because of the modulatory influence of subcortical structures activated during tonic pain. Contrary to our expectations, the combined effect of such influences increases rather than decreases NVC.

      Key Words

      Introduction

      The brain has an intrinsic mechanism of neurovascular coupling (NVC) to adjust the vascular supply to local changes in functional activity (
      • Roy C.S.
      • Sherrington C.S.
      On the regulation of the blood-supply of the brain.
      ,
      • Iadecola C.
      Neurovascular regulation in the normal brain and in Alzheimer’s disease.
      ). The understanding of NVC is fundamental to neurological sciences as well as for the proper interpretation of routinely used functional neuroimaging methods such as functional magnetic resonance imaging and positron emission tomography (
      • Hyder F.
      Dynamic imaging of brain function.
      ). Impaired NVC is believed to play an important role in several neurodegenerative diseases (
      • Lecrux C.
      • Hamel E.
      The neurovascular unit in brain function and disease.
      ).
      An understanding of NVC is far from complete, mostly because of the difficulty of direct experimental approaches. One of the indirect methods of studying NVC in human subjects is a visually evoked cerebral blood flow velocity response (VEFR). This method uses transcranial Doppler ultrasound (TCD) to measure changes in posterior cerebral artery (PCA) blood flow upon visual stimulation (
      • Aaslid R.
      Visually evoked dynamic blood flow response of the human cerebral circulation.
      ). Because of its noninvasiveness, as well as its good spatial and high temporal resolution, this method has been used repeatedly in the last decade to assess the properties of NVC in healthy subjects and in patients with suspected NVC impairment (
      • Rosengarten B.
      • Huwendiek O.
      • Kaps M.
      Neurovascular coupling and cerebral autoregulation can be described in terms of a control system.
      ,
      • Zaletel M.
      • Strucl M.
      • Rodi Z.
      • Zvan B.
      The relationship between visually evoked cerebral blood flow velocity responses and visual-evoked potentials.
      ,
      • Zaletel M.
      • Strucl M.
      • Bajrovic F.F.
      • Pogacnik T.
      Coupling between visual evoked cerebral blood flow velocity responses and visual evoked potentials in migraneurs.
      ,
      • Zaletel M.
      • Strucl M.
      • Pretnar-Oblak J.
      • Zvan B.
      Age-related changes in the relationship between visual evoked potentials and visually evoked cerebral blood flow velocity response.
      ,
      • Azevedo E.
      • Rosengarten B.
      • Santos R.
      • Freitas J.
      • Kaps M.
      Interplay of cerebral autoregulation and neurovascular coupling evaluated by functional TCD in different orthostatic conditions.
      ,
      • Olah L.
      • Raiter Y.
      • Candale C.
      • Molnar S.
      • Rosengarten B.
      • Bornstein N.M.
      • Csiba L.
      Visually evoked cerebral vasomotor response in smoking and nonsmoking young adults, investigated by functional transcranial Doppler.
      ,
      • Rosengarten B.
      • Paulsen S.
      • Burr O.
      • Kaps M.
      Neurovascular coupling in Alzheimer patients: Effect of acetylcholine-esterase inhibitors.
      ,
      • Lin W.H.
      • Hao Q.
      • Rosengarten B.
      • Leung W.H.
      • Wong K.S.
      Impaired neurovascular coupling in ischaemic stroke patients with large or small vessel disease.
      ). In these studies, VEFR was considered an indicator of the functional reactivity of the small resistance cerebral vessels that are implicated as the main effectors of the mechanism of NVC (
      • Iadecola C.
      Neurovascular regulation in the normal brain and in Alzheimer’s disease.
      ).
      It is well known that various subcortical structures play an important role in the regulation of cortical cerebrovascular reactivity (
      • Edvinsson L.
      • Hamel E.
      Perivascular nerves in brain vessels.
      ). Functional neuroimaging studies have shown that some of these structures, e.g., monoaminergic locus coeruleus (LC), are also part of the so called “pain matrix” and are thus involved in pain processing (
      • Petrovic P.
      • Petersson K.M.
      • Hansson P.
      • Ingvar M.
      Brainstem involvement in the initial response to pain.
      ). Because the visual cortex is not a part of the pain matrix per se (
      • Schweinhardt P.
      • Bushnell M.C.
      Pain imaging in health and disease—how far have we come?.
      ), it would be interesting to know whether VEFR could be affected by an ascending modulation of cerebral blood flow (CBF) and/or by cortical excitability induced by tonic pain. In this regard, a low-frequency electrical stimulation of LC has been found to cause a marked reduction in CBF in different brain areas, the effect being most evident in the occipital cortex in anesthetized cats (
      • Goadsby P.J.
      • Duckworth J.W.
      Low frequency stimulation of the locus coeruleus reduces regional cerebral blood flow in the spinalized cat.
      ). However, it is uncertain whether this was because of a simultaneous decrease in regional neuronal activity or because of a dampening effect on NVC in stress-related settings (
      • Katayama Y.
      • Ueno Y.
      • Tsukiyama T.
      • Tsubokawa T.
      Long lasting suppression of firing of cortical neurons and decrease in cortical blood flow following train pulse stimulation of the locus coeruleus in the cat.
      ,
      • Goadsby P.J.
      • Duckworth J.W.
      Low frequency stimulation of the locus coeruleus reduces regional cerebral blood flow in the spinalized cat.
      ). Nevertheless, these findings suggest that tonic pain that affects the activity of modulating intrinsic neural pathways could have a dampening effect on cerebrovascular reactivity. Therefore, in the present study we examined the hypothesis that VEFR is decreased during a tonic pain stimulus in healthy human subjects.

      Subjects and Methods

      Subjects

      Twenty-three healthy volunteers (10 males, mean age 36 ± 10 y; 13 females, mean age 38 ± 15 y) participated in the study. The study was performed with the approval of the National Medical Ethics Committee of the Republic of Slovenia. We obtained informed consent from all participants. None of the subjects had a previous history of migraine headaches or cerebrovascular or cardiovascular disease. None had been taking medications. All of the volunteers underwent a complete clinical and neurological examination as well as extracranial and TCD ultrasound to exclude any neurological or vascular abnormalities. Ultrasound examinations of the vascular status were performed using the Prosound Alpha 5SX ultrasound console (Aloka, Tokyo, Japan). We included only the subjects who had a good temporal acoustic window for the testing to ensure an artifact-free measurement throughout the entire length of one recording.

      Experimental design

      All measurements were done between 7:00 am and 10:00 am. The subjects were instructed not to drink caffeine or smoke cigarettes for at least 12 hours before the examination. The examination room was dark, quiet and warmed to an air temperature of 25°C. During the testing, the subjects were comfortably placed in a supine position on an examination table with the head and upper body elevated 45 degrees. Arterial blood pressure (ABP), heart rate (HR), end-tidal CO2 (Et-CO2) level and cerebral blood flow velocity (CBFV) were continuously measured upon repeated visual stimulation during the basal phase (400 s), during tonic pain induction (200 s) and during the recovery phase (400 s). All examinations were performed by the same examiner.
      ABP was recorded on the right hand using a Finapres 2300 blood pressure monitor (Ohmeda, Englewood, CO, USA). HR was recorded with electrocardiogram using a second standard limb lead. Et-CO2 levels were monitored through a face mask using an Oscaroxy capnometer (Datex, Helsinki, Finland). CBFV was measured using two 2-MHz transcranial Doppler ultrasound probes Delica-9 series (SMT Medical, Würzburg, Germany) attached to an individually mounted commercially available headpiece. The temporal acoustic window was used to insonate the P2 segment of the PCA on the right side and the middle cerebral artery (MCA) on the left side. Identification of the vessels followed the standard criteria described elsewhere (
      • Aaslid R.
      Cerebral hemodynamics.
      ). After the proper position was obtained, the probes were tightly fixated into place. In this way we ensured a constant angle of insonation throughout the measurement. In addition, subjects were requested to avoid head movement and speaking during the experiment.
      The visual stimulus paradigm used was a reversed-pattern checkerboard with an inverting frequency of 2 Hz presented on a 120 × 90-cm rectangular canvas placed in front of the subject at a viewing distance of 1.5 meters. A constant stimulus was used because of dependence of VEFR on stimulus features (
      • Zaletel M.
      • Zvan B.
      • Strucl M.
      • Pogacnik T.
      • Kiauta T.
      The influence of brightness, colour and complexity on visual evoked Doppler flow responses.
      ,
      • Rosengarten B.
      • Molnar S.
      • Trautmann J.
      • Kaps M.
      Simultaneous VEP and transcranial Doppler ultrasound recordings to investigate activation-flow coupling in humans.
      ). Before the start of the recording, there was a 10-min adaptation period with the visual stimulus displayed. The recording protocol consisted of 20 consecutive cycles with a resting period of 20 s (OFF period) and a stimulation period of 30 s (ON period). During the OFF period, subjects were instructed to close their eyes; during the ON period, subjects were instructed to fix their eyes on a small red cross in the middle of the canvas displaying the reversed-pattern checkerboard. Changes between periods were signaled automatically by acoustic tones, which also served as triggers for later averaging procedures.
      Tonic pain was induced by a cold pressor test (CPT), which is a standardized procedure (
      • Mitchell L.A.
      • MacDonald R.A.
      • Brodie E.E.
      Temperature and the cold pressor test.
      ). The subject’s left hand was submerged to the wrist in a plastic container filled with ice-cold water (3 to 4°C) at the beginning of the ninth OFF period and removed at the end of the twelfth ON period. The hand was then dried and wrapped with a paper towel during further recording. Subjects were asked not to move during the testing, and any additional sensory stimuli were avoided.

      Data analysis

      Analog signals were recorded by a NI USB-6215 (National Instruments Corporation, Austin, TX, USA) data acquisition card with a sampling frequency of 5 kHz. The digitalized data were sent via USB to a standard personal computer equipped with software for monitoring and storage. One recording was divided into 3 phases: the basal phase consisted of the first eight OFF/ON cycles; the CPT phase consisted of the following four OFF/ON cycles during CPT; and the recovery phase consisted of the last eight OFF/ON cycles. The mean values of ABP, HR and Et-CO2 were determined over the entire length of one OFF/ON cycle and then averaged across all cycles in each specific phase.
      An envelope curve of the CBFV signal was used to obtain peak systolic (PSV), mean (MV) and end-diastolic (EDV) velocities in the PCA and MCA. VEFR was calculated using eqn (1):
      VEFR=vSTIMvRESTvREST×100,
      (1)


      where VEFR represents the percentage of change in the PCA or MCA blood flow velocity; vSTIM the average PSV, MV or EDV during the last 10 s of ON period; and vREST the average PSV, MV or EDV during the last 5 s of the preceding OFF period. The calculation renders VEFR independent of the angle of insonation (
      • Rosengarten B.
      • Huwendiek O.
      • Kaps M.
      Neurovascular coupling and cerebral autoregulation can be described in terms of a control system.
      ). vREST was also used when comparing the resting PCA blood flow velocities between phases.

      Statistical methods

      The normality of the distribution of datasets was tested using the Kolmogorov-Smirnov test with Lilliefors correction. When a dataset showed a normal distribution, one-way repeated analysis of variance (ANOVA) measures with the Fisher post hoc test were used to compare the differences among the means of measurements in the basal, CPT and recovery phases. When a dataset showed a non-normal distribution, one-way repeated ANOVA measures on ranks with Student-Newman-Keuls post hoc test were used to compare the differences between the medians of measurements in the basal, CPT and recovery phases. Statistical significance was set at p < 0.05.

      Results

      Systemic data

      Typical recordings of all measured parameters in a single subject are shown in Figure 1, and their average values from all subjects are listed in Table 1. Both systolic and diastolic ABP significantly increased during CPT and decreased thereafter in the recovery phase, but still remained higher than in the basal phase (p < 0.05). CPT induced a statistically significant increase in HR, which was followed by a statistically significant decrease during the recovery phase compared with the basal phase values (p < 0.05). The differences in the average values of Et-CO2 between the experimental phases were not statistically significant (p > 0.05).
      Figure thumbnail gr1
      Fig. 1A typical record of a healthy subject divided into 3 phases: the basal phase consists of the first eight OFF/ON cycles (400 s) before CPT; the CPT phases are four consecutive OFF/ON cycles during CPT (each 200 s); and the recovery phase are the last eight OFF/ON cycles after CPT when the left hand is dried and wrapped in a towel (400 s). HR, heart rate; BP, blood pressure; vPCA, velocity of the blood flow in the right PCA (P2 segment); vMCA, velocity of the blood flow in the left MCA.
      Table 1Arterial blood pressure, heart rate and end-tidal CO2 data for the basal, CPT and recovery phases
      Basal phaseCPT phaseRecovery phase
      Systolic ABP (mm Hg)148 ± 19
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the CPT phase (p < 0.05).
      167 ± 21
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).
      154 ± 20
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the CPT phase (p < 0.05).
      Diastolic ABP (mm Hg)79 ± 12
      Statistically significant difference compared with the CPT phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).
      93 ± 15
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).
      83 ± 13
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the CPT phase (p < 0.05).
      HR (min−1)70 ± 11
      Statistically significant difference compared with the CPT phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).
      73 ± 12
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).
      68 ± 9
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the CPT phase (p < 0.05).
      Et-CO2 (%)5.1 ± 0.45.1 ± 0.45.1 ± 0.4
      Average mean values of the measurements are shown with corresponding SDs.
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the CPT phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).

      Resting cerebral blood flow velocity

      The average values of resting CBFV for the MCA and PCA are shown in Table 2 and Figure 2. In both MCA and PCA, there was a statistically significant increase in resting MV and EDV during CPT compared with the basal and recovery phases (p < 0.05), but no statistically significant differences between phases were detected in PSV (p > 0.05). In the PCA, there was no statistically significant variability in the resting PSV, MV or EDV between the four consecutive OFF/ON cycles during the CPT phase (p > 0.05).
      Table 2Resting PSV, MV and EDV in the MCA and PCA during the basal, CPT and recovery phases
      Basal phaseCPT phaseRecovery phase
      PCA
       PSV (cm s−1)57 ± 1057 ± 1057 ± 10
       MV (cm s−1)38 ± 7
      Statistically significant difference compared with the CPT phase (p < 0.05).
      40 ± 7
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).
      39 ± 7
      Statistically significant difference compared with the CPT phase (p < 0.05).
       EDV (cm s−1)29 ± 5
      Statistically significant difference compared with the CPT phase (p < 0.05).
      31 ± 6
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).
      30 ± 5
      Statistically significant difference compared with the CPT phase (p < 0.05).
      MCA
       PSV (cm s−1)97 ± 1898 ± 1898 ± 17
       MV (cm s−1)66 ± 13
      Statistically significant difference compared with the CPT phase (p < 0.05).
      69 ± 13
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).
      67 ± 12
      Statistically significant difference compared with the CPT phase (p < 0.05).
       EDV (cm s−1)51 ± 11
      Statistically significant difference compared with the CPT phase (p < 0.05).
      54 ± 11
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).
      51 ± 10
      Statistically significant difference compared with the CPT phase (p < 0.05).
      Average values are shown with corresponding SDs.
      Statistically significant difference compared with the basal phase (p < 0.05).
      Statistically significant difference compared with the CPT phase (p < 0.05).
      Statistically significant difference compared with the recovery phase (p < 0.05).
      Figure thumbnail gr2
      Fig. 2Scatter plot of the average mean velocity (MV) values (with corresponding SDs) during the last 5 s of the consecutive resting (OFF) periods. Both MCA and PCA values are represented.

      Visually-evoked cerebral blood flow velocity response

      In the PCA, there was no statistically significant variability in eight successive VEFRs during both the basal and recovery phases (p > 0.05). The averages of VEFR in consecutive experimental phases are shown in Figure 3. There was no statistically significant difference in peak systolic VEFR between the basal and CPT phases or between the basal and recovery phases (p > 0.05). There was a statistically significant decrease in peak systolic VEFR from the CPT phase to the recovery phase (p < 0.05). There was a statistically significant increase in mean VEFR from the basal to the CPT phase (p < 0.05) and a statistically significant decrease in mean VEFR from the CPT phase to the recovery phase (p < 0.05). There was a trend toward a decrease in mean VEFR from the basal to the recovery phase (p = 0.053). The average end-diastolic VEFR was statistically significantly different among all three phases (p < 0.05), being highest during the CPT phase and lowest during the recovery phase.
      Figure thumbnail gr3
      Fig. 3Histogram of VEFR for PSV (a) and EDV (b) in the PCA (with corresponding SDs). The VEFR is calculated as the percent increase in the average velocity during last 10 s of the ON period (visual stimulation) compared with the last 5 s of the preceding OFF period (resting). In the basal and recovery phase, eight consecutive cycles were averaged, whereas four consecutive cycles were averaged in the CPT phase. ∗ Denotes statistically significant difference between selected phases (p < 0.05).
      In the MCA, the differences in both the MV and EDV between the OFF and ON periods were statistically significant during CPT (p < 0.05) but not during the basal or recovery phases (p > 0.05). The relative increase in the MV during CPT was 1.9% and the relative increase in the EDV during CPT was 2.5%. There was no statistically significant difference between the OFF and ON periods in the PSV of the MCA during any of the phases (p > 0.05).

      Discussion

      To our knowledge, this is the first study on the effect of tonic pain induced by CPT on the VEFR of healthy subjects. We examined the hypothesis that VEFR decreases during a tonic pain stimulus. However, contrary to our expectations, we found that during CPT, end-diastolic VEFR increased, whereas peak systolic VEFR was not affected. This was accompanied by a rise in MV during the resting (OFF) period in both the PCA and the MCA because of an elevated EDV, whereas the PSV was not changed. In addition, we found that end-diastolic VEFR decreased below the basal levels in the recovery phase.
      The most important finding of our study is increased end-diastolic VEFR during CPT. In principle, increased VEFR during CPT implicates increased cerebrovascular responsiveness to visual stimulation, which could be caused by increased excitability of the cerebral cortex and/or increased activity of NVC. Previous studies with human subjects have shown that tonic pain decreases the excitability of the motor and somatosensory cortex (
      • Rossi A.
      • Decchi B.
      • Groccia V.
      • Della Volpe R.
      • Spidalieri R.
      Interactions between nociceptive and non-nociceptive afferent projections to cerebral cortex in humans.
      ,
      • Farina S.
      • Valeriani M.
      • Rosso T.
      • Aglioti S.
      • Tamburin S.
      • Fiaschi A.
      • Tinazzi M.
      Transient inhibition of the human motor cortex by capsaicin-induced pain. A study with transcranial magnetic stimulation.
      ). Similarly, in a recent study CPT reduced visually-evoked potential (VEP) amplitudes and abolished normal VEP habituation in the occipital cortex (
      • Coppola G.
      • Serrao M.
      • Curra A.
      • Di Lorenzo C.
      • Vatrika M.
      • Parisi V.
      • Pierelli F.
      Tonic pain abolishes cortical habituation of visual evoked potentials in healthy subjects.
      ), which was suggested to be caused by the modulatory action of brainstem monoaminergic nuclei during tonic pain (
      • Petrovic P.
      • Petersson K.M.
      • Hansson P.
      • Ingvar M.
      Brainstem involvement in the initial response to pain.
      ,
      • Coppola G.
      • Serrao M.
      • Curra A.
      • Di Lorenzo C.
      • Vatrika M.
      • Parisi V.
      • Pierelli F.
      Tonic pain abolishes cortical habituation of visual evoked potentials in healthy subjects.
      ). Consistent with the aforementioned studies, both noradrenergic and serotoninergic pathway stimulation has been found to have a general depressant effect on neuronal activity in various brain areas, including the visual cortex of cats and rats (
      • Sato H.
      • Fox K.
      • Daw N.W.
      Effect of electrical stimulation of locus coeruleus on the activity of neurons in the cat visual cortex.
      ,
      • Mantz J.
      • Godbout R.
      • Tassin J.P.
      • Glowinski J.
      • Thierry A.M.
      Inhibition of spontaneous and evoked unit activity in the rat medial prefrontal cortex by mesencephalic raphe nuclei.
      ,
      • Follett K.A.
      • Gebhart G.F.
      Modulation of cortical evoked potentials by stimulation of nucleus raphe magnus in rats.
      ). As shown more recently, the cholinergic nucleus basalis of Meynert (NBM), which is known to be activated during painful stimuli (
      • Zhang Y.Q.
      • Mei J.
      • Lu S.G.
      • Zhao Z.Q.
      Age-related alterations in responses of nucleus basalis magnocellularis neurons to peripheral nociceptive stimuli.
      ), exerts a depressant effect on VEPs in cats and rats (
      • Arakawa K.
      • Tobimatsu S.
      • Kato M.
      • Kobayashi T.
      Different effects of cholinergic agents on responses recorded from the cat visual cortex and lateral geniculate nucleus dorsalis.
      ,
      • Herron P.
      • Li Z.
      • Schweitzer J.B.
      Effects of cholinergic depletion on evoked activity in the cortex of young and aged rats.
      ). Therefore, the increase in VEFR during CPT in our study was probably not caused by increased excitability of the cerebral cortex, but rather because of the enhanced activity of NVC.
      Our study is the first to suggest increased NVC activity in the occipital cortex during tonic pain in humans. Previous human studies have generally shown an increase in regional CBF with noxious stimuli compared with innocuous stimuli in the areas involved in pain and somatosensory processing (
      • Casey K.L.
      • Minoshima S.
      • Morrow T.J.
      • Koeppe R.A.
      Comparison of human cerebral activation pattern during cutaneous warmth, heat pain, and deep cold pain.
      ,
      • Derbyshire S.W.
      • Jones A.K.
      Cerebral responses to a continual tonic pain stimulus measured using positron emission tomography.
      ,
      • Svensson P.
      • Johannsen P.
      • Jensen T.S.
      • Arendt-Nielsen L.
      • Nielsen J.
      • Stodkilde-Jorgensen H.
      • Gee A.D.
      • Baarsgaard Hansen S.
      • Gjedde A.
      Cerebral blood-flow changes evoked by two levels of painful heat stimulation: a positron emission tomography study in humans.
      ,
      • Moulton E.A.
      • Keaser M.L.
      • Gullapalli R.P.
      • Greenspan J.D.
      Regional intensive and temporal patterns of functional MRI activation distinguishing noxious and innocuous contact heat.
      ). However, we are not aware of any human study that under both painful and nonpainful conditions compares changes in regional CBF in areas of the cortex that are not involved in pain processing. In animals, during painful stimulation, CBF regulation is also affected in regions outside the so-called “pain matrix” (
      • Sakiyama Y.
      • Sato A.
      • Senda M.
      • Ishiwata K.
      • Toyama H.
      • Schmidt R.F.
      Positron emission tomography reveals changes in global and regional cerebral blood flow during noxious stimulation of normal and inflamed elbow joints in anesthetized cats.
      ). Interestingly, in this study the difference in CBF was largest over the posterior cortex. This effect could be explained at least in part by the activation of various subcortical structures during pain, which are known to be involved in the regulation of CBF. For example, in anesthetized rats, CBF in various cortical regions increased after the activation of NBM by focal electrical stimulation (
      • Adachi T.
      • Inanami O.
      • Ohno K.
      • Sato A.
      Responses of regional cerebral blood flow following focal electrical stimulation of the nucleus basalis of Meynert and the medial septum using the [14C]iodoantipyrine method in rats.
      ), and somatosensory evoked regional CBF was markedly attenuated when NBM was inactivated by the GABAergic agonist muscimol (
      • Piche M.
      • Uchida S.
      • Hara S.
      • Aikawa Y.
      • Hotta H.
      Modulation of somatosensory-evoked cortical blood flow changes by GABAergic inhibition of the nucleus basalis of Meynert in urethane-anaesthetized rats.
      ). The important influence of cholinergic projections on the regulation of CBF has also been demonstrated in humans in a recent study on patients with Alzheimer’s disease, in which administration of the acetylcholine-esterase inhibitor donepezil normalized the time course of the VEFR (
      • Rosengarten B.
      • Paulsen S.
      • Molnar S.
      • Kaschel R.
      • Gallhofer B.
      • Kaps M.
      Acetylcholine esterase inhibitor donepezil improves dynamic cerebrovascular regulation in Alzheimer patients.
      ). Furthermore, the increase in CBF in the MCA upon physical exercise in humans has been shown to be abolished with the cholinergic antagonist glycopyrrolate (
      • Seifert T.
      • Fisher J.P.
      • Young C.N.
      • Hartwich D.
      • Ogoh S.
      • Raven P.B.
      • Fadel P.J.
      • Secher N.H.
      Glycopyrrolate abolishes the exercise-induced increase in cerebral perfusion in humans.
      ). Therefore, the increase of the VEFR during CPT in our study suggests that the likely increase in NVC during tonic pain could be a consequence of interplay of various subcortical structures that modulate NVC in a way that favors evoked (sensory) activity. The observed small but statistically significant increase in the EDV of the MCA upon visual stimulation during CPT in our study is in line with the notion of generalized influence of tonic pain on NVC.
      Interestingly, the VEFR in the recovery phase of our study decreased to a point below the basal values. This might be viewed as a consequence of habituation to visual stimulus (
      • Sturzenegger M.
      • Newell D.W.
      • Aaslid R.
      Visually evoked blood flow response assessed by simultaneous two-channel transcranial Doppler using flow velocity averaging.
      ,
      • Niehaus L.
      • Weber U.
      • Lehmann R.
      Visually evoked blood flow responses in the posterior cerebral artery: Effects of age and stimulus conditions on the response amplitude.
      ) or a decline in the level of attention to repetitive visual stimulation (
      • Schnittger C.
      • Johannes S.
      • Munte T.F.
      Transcranial Doppler assessment of cerebral blood flow velocity during visual spatial selective attention in humans.
      ,
      • Rosengarten B.
      • Kaps M.
      A simultaneous EEG and transcranial Doppler technique to investigate the neurovascular coupling in the human visual cortex.
      ). However, a recent study showed a lack of habituation of VEP in healthy subjects lasting several minutes after ending the CPT (
      • Coppola G.
      • Serrao M.
      • Curra A.
      • Di Lorenzo C.
      • Vatrika M.
      • Parisi V.
      • Pierelli F.
      Tonic pain abolishes cortical habituation of visual evoked potentials in healthy subjects.
      ). Consistent with these findings, we observed no statistically significant variance in the eight successive VEFRs in the recovery phase of our study. In addition, pain is known to divert attention from visual processing as shown by decreased BOLD signal levels in the occipital cortex during concomitant visual and painful stimulus (
      • Bingel U.
      • Rose M.
      • Glascher J.
      • Buchel C.
      fMRI reveals how pain modulates visual object processing in the ventral visual stream.
      ). One can speculate that withdrawal of painful stimulus allows attention to be (at least partially) allocated back to the visual processing. Therefore, it is possible to assume that decrease of VEFR during the recovery phase of our study should not be solely attributed to habituation and/or loss of attention, but rather that the modulatory actions of tonic pain on the regulation of CBF and/or cortical excitability outlasted the painful stimulation. Correspondingly, a recent study has demonstrated the persistence of decreased somatosensory evoked potentials after the painful stimulus ended (
      • Fujii-Abe K.
      • Oono Y.
      • Motohashi K.
      • Fukayama H.
      • Umino M.
      Heterotopic CO2 laser stimulation inhibits tooth-related somatosensory evoked potentials.
      ). Similarly, after ending CPT in healthy human subjects, the sympathetic nerve activity of muscle is still elevated compared with the basal condition (
      • Mizushima T.
      • Tajima F.
      • Nakamura T.
      • Yamamoto M.
      • Lee K.H.
      • Ogata H.
      Muscle sympathetic nerve activity during cold pressor test in patients with cerebrovascular accidents.
      ).
      Our finding of elevated MV in the PCA and the MCA during CPT is in accord with earlier studies (
      • Roatta S.
      • Micieli G.
      • Bosone D.
      • Losano G.
      • Bini R.
      • Cavallini A.
      • Passatore M.
      Effect of generalised sympathetic activation by cold pressor test on cerebral haemodynamics in healthy humans.
      ,
      • Sohn Y.H.
      Cerebral hemodynamic changes induced by sympathetic stimulation tests.
      ,
      • Zvan B.
      • Zaletel M.
      • Pogacnik T.
      • Bajrovic F.F.
      Effect of generalized sympathetic activation by cold pressor test on cerebral hemodynamics in diabetics with autonomic dysfunction.
      ). In these studies, it was hypothesized that changes in CBFV during CPT are a consequence of an increased tone of the insonated vessels caused by a generalized activation of the sympathetic nervous system. Direct measurement of an artery diameter under the microscope during a craniotomy in humans has shown that moderate perturbations in ABP and arterial CO2 levels induce no significant changes in the outer MCA diameter (
      • Giller C.A.
      • Bowman G.
      • Dyer H.
      • Mootz L.
      • Krippner W.
      Cerebral arterial diameters during changes in blood pressure and carbon dioxide during craniotomy.
      ). In our study, the changes in ABP were in the autoregulatory range, and no changes in the Et-CO2 during CPT were detected. Therefore, we can assume that during CPT, changes in CBFV reflect changes in CBF rather than increase of vascular tone because of sympathetic activation (
      • Kontos H.A.
      Validity of cerebral arterial blood flow calculations from velocity measurements.
      ). On the other hand, changes in EDV most accurately reflect changes in the vascular tone of cortical microvessels (
      • Rosengarten B.
      • Kaps M.
      Peak systolic velocity Doppler index reflects most appropriately the dynamic time course of intact cerebral autoregulation.
      ), which are the main effectors of NVC (
      • Aaslid R.
      Cerebral hemodynamics.
      ,
      • Rosengarten B.
      • Huwendiek O.
      • Kaps M.
      Neurovascular coupling and cerebral autoregulation can be described in terms of a control system.
      ). Therefore, the increase in resting MV and EDV during CPT in our study could be caused by increased cortical activity in the vascular territory of insonated vessels. This is in accord with a study using fMRI during CPT applied to a foot that showed the anterior cingulate gyrus, superior frontal gyrus and cuneus activation (
      • Fulbright R.K.
      • Troche C.J.
      • Skudlarski P.
      • Gore J.C.
      • Wexler B.E.
      Functional MR imaging of regional brain activation associated with the affective experience of pain.
      ). Notably, in our study there was no statistically significant variance between the four consecutive OFF/ON cycles during CPT in the resting PSV, MV or EDV, suggesting a relatively fast and sustained influence of tonic pain on CBF.
      As in previous studies, we used VEFR as an indicator of cerebrovascular reactivity (
      • Rosengarten B.
      • Paulsen S.
      • Molnar S.
      • Kaschel R.
      • Gallhofer B.
      • Kaps M.
      Acetylcholine esterase inhibitor donepezil improves dynamic cerebrovascular regulation in Alzheimer patients.
      ,
      • Azevedo E.
      • Rosengarten B.
      • Santos R.
      • Freitas J.
      • Kaps M.
      Interplay of cerebral autoregulation and neurovascular coupling evaluated by functional TCD in different orthostatic conditions.
      ,
      • Olah L.
      • Raiter Y.
      • Candale C.
      • Molnar S.
      • Rosengarten B.
      • Bornstein N.M.
      • Csiba L.
      Visually evoked cerebral vasomotor response in smoking and nonsmoking young adults, investigated by functional transcranial Doppler.
      ,
      • Lin W.H.
      • Hao Q.
      • Rosengarten B.
      • Leung W.H.
      • Wong K.S.
      Impaired neurovascular coupling in ischaemic stroke patients with large or small vessel disease.
      ,
      • Yonai Y.
      • Boms N.
      • Molnar S.
      • Rosengarten B.
      • Bornstein N.M.
      • Csiba L.
      • Olah L.
      Acetazolamide-induced vasodilation does not inhibit the visually evoked flow response.
      ). However, questions remain about whether NVC can be evaluated without a simultaneous assessment of both the vascular as well as the neural response to visual stimulation (
      • Rosengarten B.
      • Molnar S.
      • Trautmann J.
      • Kaps M.
      Simultaneous VEP and transcranial Doppler ultrasound recordings to investigate activation-flow coupling in humans.
      ). Although our assumption of modifying influence of tonic pain on NVC is corroborated by a recent study using VEP (
      • Coppola G.
      • Serrao M.
      • Curra A.
      • Di Lorenzo C.
      • Vatrika M.
      • Parisi V.
      • Pierelli F.
      Tonic pain abolishes cortical habituation of visual evoked potentials in healthy subjects.
      ), a combined cerebrovascular and electrophysiologic approach could give even more insights regarding the actual mechanisms.

      Conclusion

      Our study is the first to suggest increased NVC activity in the occipital cortex during tonic pain in humans. This finding is in accordance with the modulatory influence of subcortical structures known to be activated during tonic pain. Furthermore, the increase, rather than the expected decrease, of NVC activity in the occipital cortex during a tonic pain stimulus suggests a unique interplay of various modulatory structures with opposing action on the tone of occipital cortical microvessels —dilation mediated by cholinergic projections in contrast to constriction mediated by monoaminergic projections. To further substantiate this hypothesis, additional study of the phenomena in humans and animals with the use of advanced functional imaging methods is required.

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