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RAT MODEL OF INFLUENZA-ASSOCIATED ENCEPHALOPATHY (IAE):STUDIES OF ELECTROENCEPHALOGRAM (EEG) IN VIVO

Y. CISSÉ,a S. WANG,a I. INOUEb AND H. KIDOa *a Division of Enzyme Chemistry, Institute for Enzyme Research, TheUniversity of Tokushima, Tokushima 770–8503, Japanb Division of Molecular Neurobiology, Institute for Enzyme Research,The University of Tokushima, Tokushima 770–8503, Japan

Abstract—Inuenza-associated encephalopathy (IAE) is ch-aracterized by severe neurological complications duringhigh-grade fever with high morbidity and mortality in chil-dren. The major neurological complications during high-grade fever include convulsive seizures, loss of conscious-ness, neuropsychiatric behavior (hallucination, meaninglessspeech, disorientation, laughing alone); high voltage ampli-tude slow waves and the occurrence of theta oscillation aredepicted on the electroencephalogram (EEG) in the IAE pa-tients. At the early phase of the disease, the cytokines levelsincrease in severe cases. To understand the neuronal prop-erties in the CNS leading to these neurological complicationsin IAE patients, we recorded EEG signals from the hippocam-pus and cortex of rats infected with inuenza A/WSN/33 H1N1virus (IAV) strain. Abnormal EEG activities were observed inall infected rats under anesthesia, including high voltageEEG burst amplitude and increased EEG spikes in the earlyphase (8 h–day 2) of infection, and these increases at theearly phase were in parallel witha signicant increase level of interleukin-6 (IL-6) in the serum. When the infected rats wereheat-stressed by elevating the rat body core temperature to39–41 °C, these abnormal EEG activities were enhanced, andthe oscillation pattern shifted in most of rats from slow burst-ing waves ( < 1 Hz) to theta oscillation (3–6 Hz). These resultsindicate that the abnormal EEG activities in IAE patientscould be well reproduced in anesthetized IAV infected ratsunder hyperthermia, hence this animal model will be usefulfor further understandings the mechanism of neuronal com-plications in IAE patient during high-grade fever. © 2010IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: EEG, inuenza, hyperthermia, cytokines, thetaactivity, hippocampus.

Inuenza-associated encephalopathy (IAE) is a complexinfectious disease which lead to serious neurological com-plications ( Kasai et al., 2000; Morishita et al., 2002 ) andthe levels of cytokines increase in the early phase of thedisease in some severe cases, particularly in pediatricinuenza ( Aiba et al., 2001; Fukumoto et al., 2007 ). Therates of mortality and neurological complications are par-

ticularly high in children under 5 years ( Cox and Subbarao,1999; Morishita et al., 2002 ). The clinical features of theneurological complications at high-grade fever include lossof consciousness, coma, convulsive seizures, brainedema, and multiple organs failure. In addition, some neu-ropsychiatric behaviors (e.g., hallucination, meaninglessspeech, periodic crying, and laughing alone) and at theelectroencephalogram (EEG) level, high voltage slow am-plitude oscillation and the occurrence of theta activity areoften observed at the early phase of the disease in IAE

patients ( Okumura et al., 2005; Fukumoto et al., 2007 ). Although there have been many reports on IAE in the lastdecade ( Fujimoto et al., 1998; Mori et al., 1999; Togashi etal., 2000; Chen et al., 2005 ), there is no experimentalelectrophysiological model of IAE during hyperthermia.Moreover, the EEG activities in animals infected with inu-enza A/WSN/33 H1N1 virus (IAV) during hyperthermiacondition have not been reported so far.

To design an animal model of IAE, we examined theeffects of IAV infection on EEG activities in the hippocam-pus and cortex of rats using EEG recording in vivo. Thehippocampus and the cortex were targeted for the presentmodel because these two structures are among the mostaffected areas in the brain after IAV. For example, after IAV infection in rats, the cellular proteases, such as pan-creatic ectopic trypsin and matrix metalloproteinase 9(MMP-9), are markedly upregulated in the cortex, butmainly in the hippocampus ( Le et al., 2006; Kido et al.,2007, 2008 ) and theta oscillation, which is one of the major oscillation patterns observed in IAE patients during high-grade fever is most prominent in the hippocampus and itoccurs in the cortex as well (reviewed in Stan Leung,1998 ).

In order to make the model compatible with IAE pa-tients’ physiological behavior, the experiment required tocreate hyperthermia condition. This is because rats did notmanifest spontaneous hyperthermia after IAV infection.

Hence rats were anesthetized and exposed to heat stressby raising the core body temperature to a level similar tohuman fever. Under this hyperthermia condition, the typi-cal abnormal EEG activities similar to those observed inIAE patients during high-grade fever were observed in theinfected rats. These abnormal EEG activities were in par-allel with a signicant increases level of interleukin-6 (IL-6)in the serum. It is known that infection of various types of virus including IAV induces overproduction of proinam-mation cytokines such as tumor necrosis factor alpha(TNF- ), IL-6 ( Aiba et al., 2001; Fukumoto et al., 2007 ).

This is the rst electrophysiological analysis of brainactivity in animals infected with IAV demonstrating hyper-

*Corresponding a uthor. Tel: 81-88-633-7425; fax: 81-88-633-7423.E-mail address: [email protected] (H. Kido). Abbreviations: EEG, electroencephalogram; FFT, fast Fourier trans-formation; IAE, Inuenza-associated encephalopathy; IAV, inuenza

A/WSN/33 H1N1 virus; IL-6, interleukin-6; MMP-9, matrix metallopro-teinase 9; NMDA, N -methyl- D-aspartate; PCR, polymerase chain re-action; TNF- , tumor necrosis factor alpha; 2D, two-dimension.

Neuroscience 165 (2010) 1127–1137

0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2009.10.062

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thermia-induced high voltage EEG burst amplitude slowwaves and theta oscillation, which are the major brainactivity in human during high-grade fever in IAE patients(Okumura et al., 2005; Fukumoto et al., 2007 ).

EXPERIMENTAL PROCEDURESAnimals, virus infection and electrophysiology

This study was performed in accordance with the Guidelines for Animal Care and Use approved by the animal care committee of The University of Tokushima. We used male Wistar rats weighing(80–150 g) aged 3–4 weeks (P25–P33) were used in the presentstudy. For virus infection, rats were anesthetized with ketamine– xylazine (62.6–12.4 mg/kg), then A/WSN/33 strain (1 10 5 plaqueforming unit in PBS, 60 L) was intranasally administrated. Toprevent air infection between the uninfected and infected rats, theinfected and uninfected rats were kept in two different rooms. EEGactivities in infected and uninfected rats were not recorded in thesame days and separate recording electrodes were used for infected rats and uninfected ones. The rooms were designed in

accordance with the Guidelines for Animal Care and Use of theUniversity of Tokushima.

Anesthesia and hyperthermia

Rats were anesthetized by low-dose of ether, and tted to astereotaxic frame (model SN–6N, Narishige, Tokyo, Japan). Thenthe animals were gas-anesthetized with 1.5–1.7% isouranemixed with 30% O 2 and 70% N 2 allowing spontaneous respiration.In some rats ( n 5), ketamine (62.6 mg/kg) and xylazine (12.4mg/kg) were used. The rat core body temperature was measuredbefore and after infection using digital rectal thermometer. Duringthe EEG recording, the body temperature was gradually increasedfrom 37–41 °C using a heating pad system (model 21051-00, FineScience Tools Inc., CA, USA), with a feed-back control probeinserted rectally. The rats were covered with a wool sheet (15

cm 20 cm) to reduce heat loss. The EEG recording for an ex-periment lasted for a period of 20–35 min. The slow and verypronounced EEG waves were continuously monitored during thewhole experiment to ensure that the animal was well anesthetizedand painless.

EEG recordings

To record the EEG, enamel-coated tungsten wire electrodes withuncoated diameter of 120 m (M.T. Giken Co, Tokyo, Japan)were used. Craniotomy was performed without damaging theunderlying dura using a standard miniature drill equipped with a0.5 mm diameter drill bit. The electrodes were inserted based onGeorge Paxinos and Charles Watson “The rat brain” stereotaxiccoordinates ( Paxinos and Watson, 2005 ). To insert the EEG in thehippocampal CA1–CA3 area (left hemisphere), an electrode waspositioned 2.8–3.0 mm posterior to the bregma, 2.7–2.9 mmlateral from the midline, 2.6–3.0 mm below the dura. To place theelectrode in the cortical area (right hemisphere), was positioned2.0–3.0 mm anterior from the bregma, 2.0–3.5 mm lateral fromthe midline and 0.5–0.8 mm below the dura. Signals were re-corded using a dual microprobe system (WPI Instruments, NewHaven, CN, USA), and a home-made amplier ( 1000). Thebaseline was adjusted to zero-level with a slow voltage clampsystem with a time constant of 2.2 s. The signal was low-passltered at 0.5–3 kHz, sampled at 1 kHz and recorded using

Axopatch software (Axon Instruments, Palo Alto, CA, USA). Toverify the electrode position, the electrode tip was coated with alipophilic tracer dissolved in dimethylsulfoxide at a concentrationof 1–2.5 mg/mL, before insertion into the brain. After removal of the electrodes, rats were anesthetized with ketamine–xylazine

(62.6–12.4 mg/kg) and were transcardially perfused with saline,followed by xation (4% paraformaldehyde). The brain was re-moved and immediately put in sucrose and kept in a 4 °C room.Then 800 m thick sections were prepared. Red traces left by theelectrodes with the dye were observed under the microscope andphotographed.

Four rats were used for chronically implantation EEG record-ings under non-anesthetized condition. After insertion of elec-trodes into the brain, two screws were placed on the two hemi-spheres and the skull was covered with dental cement and a boltin the cement to allow no painful xation of the rat’s head in thestereotaxic frame. After surgery, the rats were kept individually for 2–3 days for recovery. Then, the EEG signals were recorded for 1–2 days before infection as control. Then, rats were infected andEEG activities were recorded every day from 4 h to day 6 of postinfection time without heat-stressed.

Characterization of abnormal EEG signals

The EEG amplitudes, spikes, lower amplitude uctuation duringthe burst suppressed periods, and the oscillation patterns wereused to characterise abnormal events in the control (uninfected)

and infected animals. The EEG burst amplitude was measuredfrom the baseline (l 50 ms) to the peak of the burst as noted by(a–b) in Fig. 2 A and the average ( n 20 EEG bursts) was used tomake an averaged single point. The spike was identied as asharp wave. It usually sprouts randomly within the burst andduring the burst suppressed periods. To distinguish the spike fromother waves, we carefully examined the wave patterns to nd thespike has the characteristics of 1 d 15 ms and 1 k 35 ms (seeleft, Fig. 2 A).

The lower amplitude uctuation was characterised by mea-suring the sweep amplitude (h) from the peak positive to the peaknegative and 15 sweeps were taken to obtain an averaged singlepoint (Fig. 3 B, inset).

Real time-polymerase chain reaction (PCR) analysis

The total RNA was isolated from the hippocampus and cortex of infected ( n 12) and uninfected ( n 3) rats using an RNeasy Minikit (Qiagen, Valencia, CA, USA) according to the protocol suppliedby the manufacturer and then reverse transcribed using Oligoprimers and SuperScript III RT (Gibro BRL, Rockville, MD, USA)for cDNA synthesis. To determine the virus titre, forward (5 = -CAGCACTCTCGGTCTGGACAT-3 = , nucleotide 167–187), andreverse primers (5 = -TCCTTCAGAATCCGCTCCACTA-3 = , nucleo-tide 228–238) were selected from the inuenza A virus NS1 genesegment. PCR amplication consisted of 40 cycles of 15 s for denaturation at 95 °C and 1 min for extension at 58 °C. Tonormalize the quantities, mouse -actin was used for the control of RNA amplication. Quantication of gene expression was per-formed using Fast Start SYBR Green Master (Roche Diagnostics,Tokyo, Japan) with an ABI Prism 7300 system (Applied Biosys-

tems, Foster City, CA, USA). The reactions were characterized bythe point at which the PCR product was rst detected (the thresh-old cycle) instead of the amount of PCR product accumulatingafter a xed number of cycles.

Measurement of serum levels of cytokines

The serum levels of IL-6 and TNF- were measured using 15 rats,control ( n 3), and 8 h ( n 3), day 2 ( n 3), day 4 ( n 3), day 6(n 3) post infection. The EEG signals were rst recorded, thenblood was collected from the heart, and the plasma was separatedby centrifugation at 2000 g for 15 min. The serum was collectedand stored. The concentration of serum IL-6 and TNF- weremeasured according to the protocol of the immunoassay kit (In-vitrogen Corporation, CA, USA).

Y. Cissé et al. / Neuroscience 165 (2010) 1127–11371128



Statistics

Numerical values are expressed as mean SD. P -value was ob-tained by student’s paired t -test, and P 0.05 was consideredstatistically signicant.

RESULTSEEG signals in chronically implanted rats

To mimic IAE patients EEG recordings conditions, we usedchronically implanted non-anesthetize rats and tried tocreate hyperthermia condition. Inuenza virus did not in-duce spontaneous hyperthermia in the IAV infected rats.

To record EEG activities, the experiment requires stablerecordings under hyperthermia. When heat stress wasapplied, the rats responded violently moving the body con-tinuously during the heat application. Therefore the record-ing could not be realized under head-stressed condition.We could successfully record only without the heat stressat the normal body temperature in three out of four ratsexamined. In this condition, the recorded EEG activitieswere similar to that observed in normal non-anesthetizedanimal including small amplitude uctuation about 0.2 mV,irregular activities and theta oscillation dominantly at 3–6Hz. After IAV infection, the theta component increased in

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Fig. 1. Electroencephalogram (EEG) activity in chronically implanted non-anesthetize rats. (A) EEG activity in the cortex and hippocampus beforeinfection (control) and after infection at different post infection time (4 h 8 h, day 2, day 4, day 6). A section marked by the horizontal (bar and letter a–b) on the EEG traces are expanded (right). Note no striking difference between control and after infection. (B) The two-dimension fast fourier transformation (2D-FFT) displayed the frequency distribution for a period of 20 s of recording. Note an activation of theta oscillation in the hippocampuscompared to the cortex at the post infection (4 h 8 h, day 2, day 4). (C) Paroxysmal activity after infection at day 4 in both the cortex and hippocampus.Numbers (1 and 2) are expanded at the bottom. Note the presence of interictal activity and large burst in expanded traces. The 2D-FFT is calculatedfor a period of 20 s EEG activity.

Y. Cissé et al. / Neuroscience 165 (2010) 1127–1137 1129



the hippocampus compared to before infection. The two-dimension fast fourier transformation (2D-fast Fourier transformation (FFT)) displayed the increased EEG activ-

ities at theta frequency (3–6 Hz) after infection in all rats(Fig. 1 A). Paroxysmal activity was also depicted at day 4 of post infection in one out of the three infected rats ( Fig. 1 C).Hence, some variability in individual infected rats mayexist. These ndings were different from those of IAEpatients under high-grade fever. Hence, the applicationof the heat stress was necessary. To overcome thistechnical limitation, the experiments required the use of anesthesia in order to establish a stable non-movingcondition during the rat’s EEG recordings. Therefore, inthe following experiments, the EEG signals were re-corded under anesthesia during normal and heat stressconditions.

EEG signals in uninfected rats

For control experiments, we recorded EEG simultaneouslyfrom the hippocampus and cortex of uninfected rats under normal body temperature at 37 °C and under heat stressconditions at 39–41 °C. The EEG of rats at 37 °C wascharacterized by slow bursting waves ( 1 Hz) detected bythe FFT as shown in Fig. 2 B and small amplitude oscilla-tion (4–14 Hz) during the burst suppressed periods in theltered traces ( Fig. 2 A). Neuronal activities in animals dur-ing sleep were described by Steriade et al. (2001) andTimofeev et al. (2001) . Slow EEG oscillation was de-scribed in animals by Steriade et al. (1993a,b,c ) and inhumans by Achermann and Borbély (1997); Watts andHerrick (1999) and Rojas et al. (2006) demonstrated thepresence of slow oscillation under isourane anesthesia.The low amplitude uctuation during the burst suppressed

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Fig. 2. EEG recordings in control rat with or without heat stress. (A) EEG recorded simultaneously in the cortex and hippocampus. Left, top, EEG at37 °C displayed slow oscillation activity ( 1 Hz). A section of the top traces is ltered at 4 –14 Hz. Right, top, EEG recorded at 41 °C is characterizedby slow oscillation and increased burst frequency. EEG burst is indicated by the arrow–arc and spike by arrowhead. Bottom, characterization of EEGburst amplitude (left) and spike (right). (B) FFT plot shows EEG activity at 37 (left) and 41 °C (right). Note a predominant slow oscillation peaksindicated by the arrows. (C) Effect of hyperthermia on EEG burst amplitude and spikes. Note the constant EEG burst amplitude level.




periods recorded from the hippocampus was larger andclear than that from the cortex ( Fig. 2 A). Hyperthermia hadno remarkable effects as depicted on the EEG signals ( Fig.

2 A). EEG burst amplitude or number of spikes in both thecortex and hippocampus were almost the same as those atnormal body temperature ( Fig. 2 C).

Inuenza virus infection and hyperthermia induceabnormal EEG signals

We then recorded the EEG signals from infected rats. Weanalyzed the EEG burst amplitude and EEG spikes of infected rats at different post infection time, 8 h ( n 5), day1 (n 4), day 2 ( n 6), day 3 ( n 5), day 4 ( n 4), day 5(n 3) and day 6 ( n 4). The abnormal activities wereobserved from 8 h which were gradually decreased to day6 of post infection.

Fig. 3 A shows EEG activities simultaneously recordedfrom the hippocampus and the cortex at 8 h and day 2postinfection under hyperthermia (40 °C), which displayed

an enhanced low amplitude uctuation (0.1–0.5 mV) at4–14 Hz during the burst suppressed periods, high voltageEEG burst amplitude and EEG spikes compared to unin-fected rats. These abnormal activities were much pro-nounced in the hippocampus compared to the cortex ( Fig.3 A). The statistical analysis revealed that the low ampli-tude uctuation was markedly enhanced in the hip-pocampus of infected rats compared to the cortex of infected rats at 8 h and day 2 of post infection ( Fig. 3 B).The EEG burst amplitudes and the EEG spikes werealso markedly enhanced in the infected rats comparedto the uninfected. The enhancement in the hippocampus(Fig. 3 C, left) was slightly higher than in the cortex ( Fig.

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Fig. 3. Hyperthermia induces enhanced abnormal EEG activities at 8 h and day 2 of post infection in simultaneously recordings from hippocampusand cortex in inuenza A/WSN/33 H1N1 virus (IAV) infected rats. (A) EEG recorded in the hippocampus and cortex at 8 h and day 2 after infection.Left, superimposed hippocampal and cortical EEG traces at 40 °C after infection at 8 h. Note the enhanced low amplitude uctuation at 4 –14 Hz lteredfrom a period marked by the horizontal (bar and 1) on the EEG traces. The spikes are indicated by arrowhead. Right, superimposed EEG traces of the hippocampus and the cortex at 40 °C after infection at day 2. (B) The enhancement of the low amplitude uctuation in IAV infected rats duringhyperthermia (40 °C). Top (inset), the low amplitude uctuation (h) characterization. The low amplitude uctuation was signicantly enhanced in thehippocampus of infected rats compared to the cortex of infected rats at 8 h and day 2 of post infection. (C) Left, A gradual enhancement a of abnormalEEG burst amplitude (top) and increased EEG spikes (bottom) in both the hippocampus (left) and the cortex (right) of infected rats at 8 h and day 2of post infections compared to uninfected ones. All data are mean SD (* P 0.05, ** P 0.01, *** P 0.001; ( n 3: uninfected, post infection 8 h andday 2)).




3C, right). Hence, to study the effects of inuenza virusinfection during hyperthermia, we analyzed mainly theEEG recorded from the hippocampus in the subsequentexperiments.

Fig. 4 A shows representative results of the effects of heat stress on the EEG recorded from the hippocampus of rats at 8 h postinfection at different body temperatures. At

37 °C, the enhanced low amplitude uctuation at 4–14 Hzwas dominant, which is depicted in the expanded andltered trace (bar and 1). Under hyperthermia (41 °C), thepattern shifted to theta oscillation (3–6 Hz). The FFT plotsdisplay this pattern shift with a peak at 3 Hz (Fig. 4 B, Top).The 2D–FFT plots show the EEG frequency distribution atdifferent body temperatures ( Fig. 4 B, bottom). Theta oscil-

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Fig. 4. Hyperthermia induces abnormal EEG activities in rat brain infected with inuenza A virus. (A) EEG in the hippocampus recorded at differentbody temperatures (37, 38, and 41 °C). At 37 °C, the EEG pattern is characterized by slow oscillations. A segment of the activity showing enhancedlow amplitude oscillation is expanded and ltered at 4–14 Hz indicated by the horizontal bar and 1. At 38 °C, the EEG activity is characterized byincreased burst frequency. A segment of the EEG is expanded to show the spikes indicated by arrowhead. At 41 °C, the EEG pattern shifted to thetaoscillation (3–6 Hz). A segment of the activity is expanded to show the theta activity. (B) Top, FFT plot shows EEG recorded at 41 °C in uninfectedand infected rats. Note that EEG activities peaked at 0.8 Hz, indicating the predominance of slow oscillation in uninfected rat, and at 3 Hz, indicatingthe predominance of theta oscillation, in infected rat. Bottom, 2D-FFT plots. Note the irregular wide range of high frequency components in infectedrat. (C) Top, EEG activities displaying theta oscillation in the cortex and hippocampus of rat at post infection day 3. Bottom, FFT plots shows thefrequency distribution of the corresponding EEG (top) traces. Note the peak at 3 Hz. (D) Top, Effect of changes in body temperature on the number of bursts in uninfected and infected rats; middle, the increased EEG burst amplitude; bottom, increase EEG spikes for low temperature at 37compared to hyperthermia at 41 °C. All data are mean SD (* P 0.05, n 3, post infection 8 h). The 2D-FFT is calculated for a period of a minuteof EEG activity.




lation appeared under hyperthermia, but the day of ap-pearance varied between infected rats after infection; twoout of ve at 8 h, two of four at day 1, three of six at day 2,three of ve at day 3, three of four at day 4, two of three atday 5, and three of four at day 6. The theta oscillation alsoappeared in the cortex ( Fig. 4C), but none of the uninfectedrats ( n 10). Hyperthermia effect was also observed on theEEG burst frequency, the EEG burst amplitude and theEEG spikes. The number of EEG bursts increased signif-icantly with the body temperature increase and remainedalmost constant over 38 °C ( Fig. 4 D, top). The EEG burstamplitude and EEG spikes increased in parallel with therise in the rat body temperature ( Fig. 4 D, bottom). But at37 °C, the EEG burst amplitude was almost same as thatin the uninfected rats. In contrast, the number of EEGspikes increased under the same temperature. This mayindicate some difference in the neuronal mechanism lead-ing to EEG burst amplitude and EEG spikes increases.These ndings including the high voltage EEG burst am-

plitude and theta oscillation induced under hyperthermiaare similar to IAE patients EEG signals recorded duringhigh-grade fever.

Fig. 5 A shows the time-dependent changes in the EEGburst amplitude and number of spikes at two body temper-atures (37 and 40 °C) after infection. Both parametersreached peak levels during 8 h to day 2 postinfection, andthen gradually decreased to levels close to the control. Therates of these changes under the heat stress were higher than those under normal temperature.

Fig. 5 B shows the time course of inuenza virus de-tection in the brain and lung after infection. The inuenzavirus mRNA level in the lung was signicantly higher at day

2 but decreased to basal level at day 4. On the other hand,the increase in mRNA level in the brain was delayed,relative to the lung, with the peak at day 4 postinfection andreturn to basal level at day 6. Similar nding has beenreported in mice infected with IAV ( Wang et al., 2009, inpress ). Correlation between EEG abnormalities and virusdetected in the brain showed that the former appearedmuch earlier than the latter.

Effects of ketamine–xylazine anesthesia

To determine whether the hyperthermia induced abnor-mal neuronal responses in IAV infected rats were de-pendent on the anesthesia, we used ketamine–xylazine

anesthesia under heat-stressed condition. It is knownthat ketamine–xylazine anesthesia induces slow sleeposcillation ( 1 Hz) without generating theta oscillation inanimal experiments ( Steriade et al., 1993a; Timofeev etal., 1996 ). However, as shown in Fig. 7 A, the EEGactivities in the IAV infected rats recorded under ket-amine–xylazine anesthesia displayed theta oscillationsimilar to that found under isourane anesthesia ( Fig.7 A). In contrast, the EEG burst amplitude and the num-ber of spike decreased compared with those observedunder isourane anesthesia ( Fig. 7 B). These ndingssuggest that the appearance of theta oscillation, highvoltage EEG burst amplitude and increased EEG spikes

Fig. 5. Hyperthermia increases EEG burst amplitude and number of spikes in rats infected with inuenza A virus. (A) EEG burst amplitude(top) and number of spikes (bottom) plotted against time after infec-tion. Note the EEG burst amplitude and number of spikes reachedpeak values at 8 h–day 2 postinfection (* P 0.05, n 3). comparinginfected rats at 37 to at 40 °C. Note the increased EEG spikes at 37 °C.(B) Changes in viral RNA in the brain and lung after intranasal instil-lation of the virus. Viral RNA was undetected at 8 h and reached apeak at day 2 in the lung and at day 4 in the brain after intranasalinfection. C stands for the uninfected (control) rats. All data aremean SD, points at 37 and 40 °C for the same post infection time arestatistically compared (* P 0.05, n 3). For interpretation of the refer-ences to color in this gure legend, the reader is referred to the Webversion of this article.




were heat-stress related events, but may also suggestthese abnormal EEG activities may be induced by dif-ferent neuronal mechanism.

DISCUSSIONWe have shown that in inuenza-infected rats, in the ab-sence of heat stress, the EEG signals did not displayabnormal neuronal responses. However, under anesthesiaand during hyperthermia condition, EEGs displayed high

voltage EEG burst amplitudes, increased EEG spikes, theEEG pattern shift from slow waves to theta oscillation.These abnormalities reached the peak levels at 8 h to day2 of postinfection and were enhanced under hyperthermia.The major nding in the present study is that the abnormalEEG activities (i.e high voltage EEG burst amplitude slowwaves and theta oscillation) in IAE patients could be wellreproduced in anesthetized IAV infected rats under hy-perthermia.

The cytokines productions are usually associated withneurological complications in IAE patients. We measuredthe amounts of IL-6 and TNF- in the serum after mea-surements of EEG activities under hyperthermia ( n 15).

The increases in the serum IL-6 levels were statisticallysignicant at the early phase of post infection (8 h–day 2)(Fig. 6 A). But the TNF- level was lower even undetect-able in some rats. The IL-6 level correlate with the abnor-mal EEG activities during hyperthermia in infected rats(Fig. 6 B). IL-6 is used in clinics as one of the diagnosticparameter in IAE patients ( Aiba et al., 2001; Fukumoto etal., 2007 ). Its high concentration was associated with neu-rological complications in IAE patients and once the serumIL-6 level was increased to 15,000 pg/mL none of thepatients survived. High IL-6 levels have also been mea-sured in mice infected with IAV (data not shown).

The hyperthermia condition was the most critical factor to aggravate the abnormal EEG activities in IAV infectedanimals. This was clear in the chronically implanted non-anesthetized rats without heat stress ( Fig. 1 A). The EEG inthese rats at normal body temperature displayed patternscommonly observed in rats during wakefulness, character-ized by small amplitude, irregular activities and theta os-cillation in the EEG of the cortex or hippocampus. But thetheta was dominant in the hippocampus after infection.These characteristics are similar and common to other mammals studied to date ( Nita et al., 2008; Steriade andTimofeev, 2003 ) including humans ( Arnolds et al., 1980 ).

Two distinct rhythmic activities depicted on the EEGwere prominent and consistently present in the infectedrats during hyperthermia condition under the anesthesia.

These rhythmic activities were (1) theta (3–6 Hz) oscilla-tion, and (2) low amplitude uctuation (4–14 Hz) during theburst suppressed periods.

It is known that theta oscillation in the hippocampus ismost consistently present during exploration ( Jouvet,1969; Vanderwolf, 1969; Winson, 1972; Buzsaki, 2002 )and rapid eye movement (REM) sleep ( Vanderwolf, 1969 )in non-anesthetized animals. In clinic, the EEG of IAEpatients with inuenza is characterized by high voltagetheta oscillation during high-grade fever ( Okumura et al.,2005; Fukumoto et al., 2007 ). Our nding is the rst ex-perimental evidence to show theta oscillation during sleep(slow wave) oscillation and under anesthesia with both

Fig. 6. Serum cytokines levels and EEG signals at different postinfection time. (A) The serum interleukin-6 (IL-6) level was high at theearly phase of post infection (8 h–day 2). (B) EEG recording in thehippocampus of the same rats at different post infection time. Notehigh voltage EEG burst amplitude at post infection time (8 h–day 2). Alldata are mean SD, (* P 0.05, n 3).




isourane and ketamine–xylazine in the infected rats dur-ing hyperthermia.

The pattern shift to theta may explain in part the occa-sional shift of neuronal activity in the brains of some pa-tients infected with inuenza virus during high-grade fever from a normal to a hyperactive brain state, similar to theawake or REM state. Therefore, we assume this patternshift leading to an unbalance in the neuronal activity is the

most sensitive period for patients to undergo abnormalbehavior changes during sleep.The low amplitude uctuation (4–14 Hz) during the

burst suppressed periods was spontaneous and present inboth uninfected and infected rats under anesthesia. Thisrhythmic activity was slightly reected in the cortex (seeltered traces, Figs. 2 – 3 A). It was clear but lower ampli-tude in the hippocampus of control animals, even morepronounced in the hippocampus of infected animals ( Fig.3 A, B) and was observed in all infected rats (8 h– day 6).This may suggests a possible involvement of other brainstructures within a large neuronal network in the genera-tion of this rhythmic activity mainly present in the hip-pocampus. It is known that hippocampus receives rhyth-mic inputs from many brain areas including the medialseptum and diagonal band complex (MS–DB) ( Chandler and Crutcher, 1983; Amaral and Kurz, 1985; Manseau etal., 2008 ), the entorhinal cortex (perforant path axons)(Bliss and Lomo, 1973 ) and the suppramammillary nuclei(Vertes and Kocsis, 1997 ). The MS–DB is believed to actas a pacemaker for the hippocampal theta generation(Goutagny et al., 2008 ). Thus, the interaction betweenthese inputs may play a role in the generation and thespread of the rhythmic low amplitude uctuation (4–14 Hz)in the hippocampus and the cortex of rats.

The factors that triggered the generation of the highvoltage EEG burst amplitude and the increased EEG spike

in IAV infected rats during hyperthermia and their physio-logical meaning are not clear. Our experiments show thatthese abnormal EEG activities under the ketamine–xyla-zine anesthesia were less than those under the isouraneanesthesia ( Fig. 7 ). Ketamine is an antagonist of N -methyl-D-aspartate (NMDA) receptor ( Brooks et al., 1997; Cisse etal., 2004; Hetman and Kharebava, 2006 ) and also knownto perform as useful analgesics. Ketamine attenuates neu-

ropathic pain in association with decreased EEG amplitudeand cortical somatosensory evoked potential amplitude(Kochs et al., 1996 ; Oga et al., 2002 ). And also it exhibitsa neuroprotective effect in inammatory pain in neonatalrodents ( Anand et al., 2007 ). Based on these reports, IAVinfection that triggers abnormal biochemical signals may ex-cessively stimulate NMDA receptors during hyperthermia.

In addition, we observed that intra-peritoneal adminis-tration of picrotoxin (5 mg/kg) known as GABA receptorsblocker ( Yoon et al., 1993 ) did not decrease the EEG burstamplitude or EEG spikes in the infected rats (data notshown). Also another factor to be considered is that isou-rane blocks gap junctions ( Peracchia, 1991 ), which formcontacts between inhibitory interneurons. This likely re-duces synchrony between interneurons, and, therefore,may affect excitatory interactions with overall result of increased EEG burst amplitude. Hence, we assume thatthe high voltage EEG burst amplitude and increased EEGspikes are, at least in part, related to the activation of NMDA receptors during hyperthermia. However, themechanism underlying the activation of the NMDA recep-tors is unknown.

Acknowledgments—This work was supported in part by Grants-in-Aid 20611013 and for The Special Coordination Funds for Promoting Science and Technology of Ministry of Education, Cul-ture, Sports, Science and Technology of Japan.

Fig. 7. A decrease of number of spikes and EEG burst amplitude in infected rats under ketamine–xylazine anesthesia. (A) EEG recorded over thehippocampus in rats at different body temperatures (37, 38, and 41 °C). The EEG activity is characterized by slow oscillations at 37–38 °C and thetaoscillation at 41 °C. Bottom, segment indicated by (1, 2, 3 and horizontal bar) on each EEG trace is expanded and ltered. (B) Effect of hyperthermiaand type of anesthesia on EEG burst amplitude and spikes. Note the reduced EEG burst amplitude and number of spikes in rats anesthetized withketamine–xylazine compared with those with isourane anesthesia. Data are mean SD, ( n 3).




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(Accepted 29 October 2009)(Available online 3 November 2009)


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