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Histamine at high dilution reduces spectral density in delta band in sleeping rats

Homeopathy (2005) 94, 86–91 r 2005 The Faculty of Homeopathy
doi:10.1016/j.homp.2004.10.002, available online at http://www.sciencedirect.com
ORIGINAL PAPER
Histamine at high dilution reduces spectral density in delta band in sleeping rats
G Ruiz-Vega1,􏰍, B Poitevin2 and L Pe ́ rez-Ordaz1
1Laboratorio de Biofı ́sica, Instituto de Fı ́sica y Matema ́ticas, Universidad Michoacana de San Nicola ́s de Hidalgo, Morelia, Michoaca ́n, Me ́xico
2Association Franc-aise por la Recherche en Homeopathie
Histamine is a central neurotransmitter, it increases arousal via H1 receptors. This study examines the effect of ultra-diluted histamine on arousal through changes in the sleep pattern of Wistar rats. The spectral density in delta (0.5–2.5 Hz) band, one of the three major spectral components of the sleep-electroencephalogram, was analyzed against time. Rats were randomized to receive histamine 30c (histamine 30c, 0.05ml every 20min during the first 2h orally), histamine intraperitoneal pre-treatment/histamine 30c (histamine 6 mg/kg i.p., followed by histamine 30c) or solvent control. The mean delta band spectral density was lower in the histamine 30c and histamine pretreat- ment/histamine 30c groups than the control group. Significant differences between histamine 30c and baseline during the first 2 h imply an immediate effect. These results also suggest a dynamic process in which the system spontaneously evolves between two locally stationary states according to a power law. From the time perspective, the system approaches, asymptotically, an equifinal state. Homeopathy (2005) 94, 86–91.
Keywords: histamine; ultra-diluted; homeostatic; self-response; sleep pattern; homeopathy
increase wakefulness and decrease slow wave sleep;
while H3 receptors play an active part in these mechanisms by regulating histamine release.11,12
As a highly diluted stimulus applied during the sleep cycle of healthy subjects, histamine would be expected to increase arousal, according to the similia principle; which in turn promoting a homeostatic host response. Sleep loss, either pharmacological or mechanical, provokes a counteraction, called ‘sleep homeostasis’, which involves an increase in non-REM sleep intensity. The main marker of the intensity of non-REM sleep is EEG slow-wave activity, defined as the spectral density in delta band, which normally declines in the course of sleep time and increases in the sleep deprivation.13 In this study, we evaluated sleep intensity as the spectral density in 0.5–2.5Hz band, calculated over the completenoise-freefile.Thisassumptionreliesonthe fact that this domain constitutes the main frequency of non-REM sleep.14 It exceeds those of waking and rapid-eye-movement (REM) sleep by an order of magnitude,15 and closely correlates to slow wave non-REM activity. This marker has been described in previouspapers.16–18
We assessed the effect ultra-low-dose (ULD) hista- mine on arousal using this parameter. In addition, as the effect of the combination of pharmacological and ultra-low-dose of the same compound has been formerly reported previously,18–20 we also investigated the combination of pharmacological+ULD histamine.
Materials and methods
Animals
Young adult male Wistar rats weighing between 250 and 350 g ðn 1⁄4 36Þ; were used. All procedures complied withMexicanFederalRegulationsfortheUseandCare of Laboratory Animals (NOM-062-ZOO-1999). Rats were feed ad libitum on standard diet (Purina, Mexico) and randomized in three groups treated as follows:
􏰐 Solvent (orally),
􏰐 Histamine 30c (orally),
􏰐 Histamine pre-treatment/Histamine 30c: histamine
intraperitoneally (6 mg/kg i.p.) followed by histamine 30c orally.
The oral dosage was 0.05 ml each 20 min during the first 2h. Pre-treatment was one i.p. injection of histamine (histamine dihydrochloride) in double dis- tilled water.
Rats were anaesthetized (40mg/kg pentobarbital), three 1.5mm diameter stainless-steel electrodes (resis- tanceo0.1 O) were implanted in the animals’ cranium after trepanation with a hand-held Foredom drill, securedwithdentalresinandconnectedtoa15A54 Grassmultichannelamplifier(Astro-Med,Inc.West Warwick, RI, USA). A bipolar EEG was recorded through two of these electrodes, which were implanted bilaterally in the parietal region, and the third one in
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Biological effect of highly diluted histamine
G Ruiz-Vega et al
the frontal area for reference (ground). Trepanation points were spotted stereotaxically at the following coordinates: 3.8 mm posterior to Bregma and 4.5 mm lateral to the side of central line for the bilateral electrodes.21 After surgery the animals were allowed to recover for 6 days; a further 1-day habituation period took place, in which the electrodes were connected to the signal amplifier by a cable that allowed freedom of movement. During the pre-operation and recovery periods, rats were maintained under a steady light–- dark cycle. Next day baseline was recorded without any mechanical or pharmacological disturbance. The next day treatment measurements started.
Sleep deprivation could be the consequence of pharmacological or mechanical stimuli (the repetitive sleepdisturbancefororaltreatmenteach20minduring 2 h); or superimposed effect of both. Hence, in order to estimate objectively the possible effect of the highly diluted histamine in histamine pre-treatment/ hista- mine30cgroupparalleltestswerecarriedoutwithtwo additional groups (12 animals per group): Histamine i.p.(6mg/kg,i.p.)andsaline,withnosleepdisturbance due to repetitive oral administration. These were supplementary ‘positive controls’.
Materials
Histamine hydrochloride was obtained from Sigma Chemical Co. (St Louis, MO, USA). Saline solution (0.9% NaCl) was obtained from PiSA, Laboratorios Mexicanos. Ultra-high diluted histamine and the solvent (87% alcohol/water) were acquired from Laboratorios Medicor (Mexico). Preparation of the histamine 30c involved 30 successive centesimal dilu- tions (1/99 vol%). The final concentration is theoreti- cally equivalent to 0.9 􏰑 10􏰏61 M.
Experimental set-up
Rats were housed individually in wooden cages built to avoid external disturbances but allowing observa- tion. Daylight was provided so as not to alter the circadianhistaminepattern.22Eachsubjectwascon- nected to a 15A54 Grass amplifier module, its controls set as follows: sensitivity 20.0mV/div, display gain 1, band-pass filter 0.3–30Hz, line filter on. The output wassampledat8Hzandonlinedigitizedwitha National Instruments AT-MIO-64E-3 card and Poly- Views v 2. software (Astro-Med). Output was stored in a computer for mathematical analysis. Sampling frequency was chosen taking into account delta band features14 as well as the Nyquist theorem.23
Experimental procedure
Six rats were tested at a time, one to a cage, two for eachgroup(fortechnicalreasons,1sessionwasrun withonly5subjects).Measurementsstartedtheday after baseline recording . At this time rats were fed their regular diet and purified water ad libitum. At 10.00, t 1⁄4 0 (t, time in hours), rats from histamine
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G Ruiz-Vega et al
pre-treatment/ histamine 30c group received a single dose of histamine i.p., simultaneously oral treatment was initiated for all three groups. On the completion of 8h recording session, the tests concluded for the six subjects and the process was repeated with six new ones until all were tested. Mathematical analysis of datawascarriedout‘blind’.Eachrecordwassplitinto 1-hperiodsandepisodescontainingEEGartefacts (disconnections or body movement) were visually identified (blind) and omitted from further analysis. Becauseofexcessivenoisyevents,oneanimalwas discarded in histamine 30c group. The 280 series of 1 h data(35ratsx8hperrat)weresubjectedtoafast fourier transform (FFT) algorithm and the spectral density was computed for the specified bands in every file (STATISTICAs, StatSoft, Tulsa, OK, USA). Due to different lengths of noisy periods, the records were of different sizes. In order to have a common reference, the spectral density in the delta band was analyzed as percentage of the spectral density in the whole frequency range (0–2.5 Hz).
Analysis of data
Results arose from a between–groups and repeated measures approach, comparing the mean value in the groups, but considering their own baseline record as
SOLVENT
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90 90 88 88 86 86 84 84 82 82 80 80 78 78 76 76 74 74 72 72 70 70 68 68
(a) HISTAMINE PRETREATMENT/HISTAMINE 30c (b)
90
88
86
84ec ddd84
c c
d
82 d 80
78
76
74 72 70 68 (c)
c
ecd
90 88 86
82 80 78 76 74 72 70 68 (d)
BASELINE AND POSITIVE ip CONTROL
BASELINE
SALINE ip
Histamine ip
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Figure 1. Variation for spectral density in delta band in all groups. Mean spectral density, sd vs time, t. sd (%) in y-axis, time in hours in the x-axis from t 1⁄4 0 to 8. Each value is mean 7 SEM in (a–c). A similar pattern is seen in (a–c); histamine 30c exhibits, at t 1⁄4 1; 2 the lowest value as well as the greatest increase rate in the middle stage. As time goes on, all groups tend to baseline state. In (d), baseline exhibits a moderate enhancement up to t 1⁄4 5; a slight decrease tendency is developed after that, as expected in the absence of sleep disturbance. A similar pattern is observed for all ‘positive’ controls, which overlapped. aDifferent from baseline, Po0:01; bPo0.05. cDifferent from solvent, Po0:01; dPo0.05. eDifferent from histamine 30c, Po0:01:
well. Comparison between-groups was by ANOVA, specifying the repeated measures (ie baseline/stimulus) as the within-subject factor. A multiple comparison procedure, the Newman–Keuls post-hoc test, was performed to determine significant differences between pairs of the three experimental groups. To avoid bias duetoextremevalues,outliersthatwereoutsidethe rangeof72sd,wereidentifiedandexcluded:3,2and 1 in solvent, histamine 30c and histamine pre- treatment/ histamine 30c groups respectively. Statis- ticalanalysiswascarriedoutwithSTATISTICAs.
Results
1. Solvent orally: Sleep disturbance due to oral dosing was seen, but no other significant difference from baseline was measured. In fact, the spectral density in delta band increases smoothly from a minimum at t 1⁄4 1 (t 1⁄4 time in hours), due to repetitive awakenings for oral administration, to a maximum at t 1⁄4 5; a small decrease was observed during the following 3 h, as expected from the circadian pattern24 (Figure 1a).
2. Histamine 30c orally: Spectral density in the delta band was less than solvent through the whole
HISTAMINE 30c
c
a b
c
d
d
d
recording period, the difference was highly significant ðPo0:01Þ during the first 4 h. However, if was different from baseline only in the first 2 h (Figure 1b).
3. Histamine pre-treatment/ histamine 30c: This group was significantly different from solvent through- out , but there was no difference from baseline at any time. The difference from the histamine 30c group was significant only in the first 2 h (Figure 1c).
4. Positive controls: histamine and saline i.p.: Baseline recordings showed a slight increase in delta spectral densityatt1⁄42;afterwards,asmoothtendencyto decline was observed. A similar pattern was seen for the positive controls, no differences from baseline were detected at any time. Since delta activity subsides spontaneously from the first to the second non-REM stage in absence of sleep deprivation24 the baseline pattern is as expected outcome when there is no sleep loss. The ‘positive’ control (histamine i.p. only) results were as expected, since i.p. histamine does not cross the blood brain barrier (Figure 1d).25,26
Three time periods were identified:
1. t 1⁄4 022: Oral dosing was given in
period. Highly significant differences ðPo0:01Þ; were found for histamine 30c compared to both baseline and solvent. The pre-treated group was not different from its baseline. Solvent values were approximately con- stant.
2. t 1⁄4 225: During this stage the host response to sleep deprivation was observed; a continuous increase in the spectral density in delta band was observed for the two histamine 30c groups but not in positive controls (which has not suffered sleep deprivation due to dosing) (Figure 1d). A linear relationship between log(spectral density, sd) vs log(time, t) was found. Figure 2 shows the fitting parameters and the correla- tion between experimental data (full line) and the linear model (dashed line). The function is presented as the general form
log y1⁄4bþm log x,
Histamine 30c PreTreat/Hist 30c Solvent
4.50 4.46 4.42 4.38 4.34 4.30 4.26
0.6 0.8
1.0 1.2
log (time, t)
Solvent: log(sd) = 4.41 + 0.04*log(t), R = 99.4% Hist pre-treat: log(sd) = 4.32 + 0.06*log(t), R = 99.3% HIstamine 30c: log(sd) = 4.19 + 0.15*log(t), R = 97.9%
Solvent
Hist pre-treat/histamine 30
Histamine 30c
1.4 1.6 1.8
Figure 2. Log–log fitting for mean spectral density in delta band. log (sd) vs log (t) from t 1⁄4 2 to 5. Linear fitting was chosen because of its high correlation, R. Fitting function as dashed line.
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this time
0
2345 2345 2345
Figure 3. Rate of change for the spectral density in delta band. Time in hours in the x-axis. Values of histamine 30c group and pre-treated group are approximately 3.5 and 1.5 times those of solvent respectively. There is a decreasing trend from maximum at t 1⁄4 2; ie, immediately after the end of dosing. The changes evaluated are d(sd) during a time interval dt in Eq. (1) for each group.
where y1⁄4sd; x1⁄4t; m1⁄4slope, b1⁄4intercept with y-axis.
The Quasi-Newton method was employed in the regression parameters estimate, calculations were performedwithSTATISTICAs.Thisrelationcanbe changed to the form
y 1⁄4 Cxa (1) where
C 1⁄4 ExpðbÞ;a 1⁄4 m.
The change rate, in this interval, was also calculated from dy/dt in the Eq. (1) for each group. Changes in the stimulated groups were different from those of solvent. These results are shown in Figure 3 where a gradual decrement is observed in all groups. The rate for histamine 30c was the greatest; it was approxi- mately 3.5 times faster than solvent; in contrast, histamine pre-treatment/ histamine 30c group rate was E1.4 times faster than solvent.
3. t 1⁄4 528: The rising spectral density seen in the precedingperiodstopped;insteadasmoothdeclining tendency begun. No difference from baseline was identified for histamine 30c and histamine pre-treat- ment/ histamine 30c groups, but they remained significantly different from solvent control.
Discussion
The statistical analysis compared the values of treatmentgroupswiththoseofsolventcontroland their own pretreatment baseline. The three treatment groups were under similar experimental conditions, ie., repetitive sleep disturbance for oral dosing (each 20min for the first 2h). Hence, it was expected that three groups developed a similar trend in time, which in fact they did (Figure 1). The consistency of the solvent group through the whole period implies the lack of treatment effect: the values were similar to
Biological effect of highly diluted histamine
G Ruiz-Vega et al 6
5
4
3
2
1
89
log (spectral density, sd)
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baseline. These results confirm the accuracy of the selected marker.
The histamine 30c results demonstrate that ULD histamine modifies the sleep pattern; the decrease in spectral density during the first 2h in histamine 30c group agrees with the experimental hypothesis suggest- ing an increase in wakefulness. The sleep pattern is characterized by three major rhythms: delta, slow (E0.3Hz) and spin spindle rhythms;27 however, the spectral density in delta band characterizes the system behavior under sleep perturbation.
The histamine pre-treated (6 mg/kg i.p.) group also exhibited an initial decrement in the spectral density, however in contrast with histamine 30c it was not different from baseline. In order not to overestimate the possible effect of the highly diluted histamine in these rats, additional ‘positive’ controls were included (histamine 6mg/kg i.p. and saline i.p.). This was to control for the effect of histamine i.p. alone without any sleep disturbance due to oral administration. These results confirm the accuracy of the outcome measuresincesleeplosswasnotdetectedwithi.p. histamine (Figure 1d); this conforms with the conven- tional blood brain barrier model.25,26
The experimental results with the administration of the combination of two doses19,20 ie. pharmacological/ ULD doses, suggest that the ULD dose neutralizes the action of the substantial dose. However, i.p. histamine alone had no effect (Figure 1d). We cannot claim neutralization of non-existent effect! It would be possible to suggest that pharmacological dose has inhibited the response to the ultra-low-dose stimulus. Additional work is required to address this issue.
TheEq.(1),y1⁄4Cxa;correspondstothegeneral
form of the mathematical relations known as power
laws; their main feature is self-similarity or scale
invariance,28inturnassociatedwithfractality.The
fractal concept was coined to describe irregular
geometric forms that lack a characteristic (single) scale
of length, but can also be applied to self-similar
phenomena evolving in time.29,30 The expression self-
organized critically (SOC) has been introduced,31 to
describe the tendency of dissipative systems to drive
themselves to a critical state, which is identified as an
attractor for the dynamics. Power laws are ubiquitous
in nature; they have been identified in diverse natural
phenomena including the restoring force in a linear
spring, Newton’s law of gravity, membrane channel
openings, human writing, biological evolution, eco-
nomics, music, earthquake structure, etc., which
suggest that SOC might be an universal phenomen- on.28,29
The three experimental groups show a SOC tendency. They evolved between two different homeo- staticstates,bothofthemattainedspontaneouslyand conventionally recognized:32
(1) The conscious state of quiet wakefulness through the first 2h when the subject was disturbed by dosing
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every 20min; hence, the sleep cycle was prevented from evolving in spite of the animal being at the beginning of its circadian sleep cycle.
(2) The unconscious stable state of NREM sleep in the last 3 h, which is asymptotically reached. Because of this feature, it is identified as a globally stable and attractive state.
After the stimulus administration, the system spon- taneously evolved to NREM sleep, characterized by the dominance of delta rhythm. The immediate response system, between t 1⁄4 2 and 5, evolved accord- ing to a power function. The periods of transition from an active to a resting state are described by an asymptotic decline of the variable (metabolic rate, breathing rate and tidal volume), such asymptotic decrease is characterized by the change of rate which gradually evolves from a maximum to a minimum when the system approaches to the lowest value and reaches the resting state.32
In our case, the variable does not decline, because deltaoscillationsarethemainfeatureofNREMsleep, its prevalence (expressed as the percentage) in contrast, increases. However, as time goes by, the rate of change diminished as it gets closer to its final value (Figure 3). Because the rate of change in the histamine 30c group was significantly higher than solvent, a synergic effect of the ultra-low-dose stimulus may be suggested.
In addition, when certain final state is specific for a given system in determined circumstances, no matter the initial conditions, the tendency to reach such a steady state is termed ‘equifinality’.32 Because from different initial conditions the three experimental groupsevolvedtoreachthesameequifinalstate(ie, no difference from baseline), it is assumed that the system has been self-organized in time to reach a global attractor,whichinturnimpliesfractality.Theadapt- ability of response is crucial to confer robustness under unexpected circumstances. Robustness and fractality are generic characteristics of SOC,33 it is not unreason- able to think of homeostasis as a fractal process for restoring the dynamics of health.
The concept of fractal physiology30 as adaptive processes which guarantee to respond to unpredictable stimuli and stresses is widely accepted. The high correlation between our experimental data and the SOC model suggests that the system has evolved from one state to another one according to a power law.
Conclusion
These results suggest that histamine 30c induced not only a transient sleep loss, reflected by the initial decreaseinthespectraldensityindeltaband,butalsoa synergic effect on the immediate host response. A self- similar pattern was identified after dosing: the system evolved driven by a threshold dynamics between two critical states; which implies the organism was impelled
Homeopathy
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to leave its initial stationary state because of the ULD histamine.
Acknowledgements
This research was supported by a grant from ScientistResearchCouncilfromUniversityofMi- choacan. We thank Rafael Villalobos-Molina and Daniel Garcı ́a-Garrido.
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