For Italy:

"It is declared that this product, brand JVC, conforms to the Ministry Decree n. 548 of 28 Aug.'95 published in the Official Gazette of the Italian Republic n. 301 of 28 Dec.'95"

CAUTION

Cassettes marked "D-VHS", "S-VHS" and "VHS" can be used with this video cassette recorder. However, D-VHS recordings are possible only with cassettes marked "D-VHS". D-VHS is a new digital memory system that uses D-VHS tapes. D-VHS was developed as a memory system for multimedia applications that require storage for large volumes of information, such as for digital video. SHOWVIEW is a registered trademark of Gemstar Development Corporation. The SHOWVIEW system is manufactured under licence from Gemstar Development Corporation.
To avoid electric shock or damage to the unit, first firmly insert the small end of the mains power cord into the recorder until it is no longer wobbly, and then plug the larger end of the mains power cord into a mains outlet. When you are not using the recorder for a long period of time, it is recommended that you disconnect the power cord from the mains outlet. Dangerous voltage inside. Refer internal servicing to qualified service personnel. To prevent electric shock or fire hazard, remove the power cord from the mains outlet prior to connecting or disconnecting any signal lead or aerial.
The STANDBY/ON current on and off. " button does not completely shut off mains power from the unit, but switches operating " shows electrical power standby and " " shows ON.
Video tapes recorded with this video recorder in the LP (Long Play) mode cannot be played back on a singlespeed video recorder.
Failure to heed the following precautions may result in damage to the recorder, remote control or video cassette.
1. DO NOT place the recorder. in an environment prone to extreme temperatures or humidity. in direct sunlight. in a dusty environment. in an environment where strong magnetic fields are generated. on a surface that is unstable or subject to vibration. 2. DO NOT block the recorders ventilation openings. 3. DO NOT place heavy objects on the recorder or remote control. 4. DO NOT place anything which might spill on top of the recorder or remote control. 5. AVOID violent shocks to the recorder during transport.

MOISTURE CONDENSATION

Moisture in the air will condense on the recorder when you move it from a cold place to a warm place, or under extremely humid conditionsjust as water droplets form in the surface of a glass filled with cold liquid. Moisture condensation on the head drum will cause damage to the tape. In conditions where condensation may occur, keep the recorder turned on for a few hours to let the moisture dry.

ABOUT HEAD CLEANING

After an extended period of use, the video heads can become dirty, resulting in a loss of picture or sound during playback. If this happens, clean the video heads by using the optional cleaning tapes.
Symptoms of dirty video heads: The picture is not clear, or does not appear. There is no sound. Mosaic (block) noise appears in the picture. Black or mosaic horizontal stripes appear in the picture. The picture stops (as if the tape is paused). A blank black or blue screen appears. The picture is fuzzy. (VHS playback)

VHS playback

Early symptom D-VHS playback

Late symptom

Block noise

Still image

Black screen
Use a cleaning tape designed specifically for D-VHS video heads (JVC D-VHS video head cleaner DFC-2) to clean the video heads. In order to avoid misoperation, set "NAVIGATION" to "OFF" ( pg. 39). Follow the instructions that are provided with the cleaning tape. If you still do not get a clear picture after using a cleaning tape: The heads may be worn. Contact your nearest JVC dealer. During VHS playback, if there is a tracking problem, the picture may appear fuzzy. Adjust the tracking manually ( pg. 17).
INSTALLING YOUR NEW RECORDER
It's essential that your video recorder be properly connected. Follow these steps carefully. THESE STEPS MUST BE COMPLETED BEFORE ANY VIDEO OPERATION CAN BE PERFORMED.

Basic Connection

Aerial terminal Back of TV

CHECK CONTENTS

Make sure the package contains all of the accessories listed in "Specifications" ( pg. 82).

SITUATE RECORDER

Place the recorder on a stable, horizontal surface.

CONNECT RECORDER TO TV

a Disconnect the TV aerial cable from the TV. b Connect the TV aerial cable to the ANT. IN jack on the rear panel of the recorder. c Connect the provided RF cable between the ANT. OUT jack on the rear panel of the recorder and the TVs aerial terminal. d Connect the provided SCART cable between the AV1 IN/OUT socket on the rear panel of the recorder and the TVs 21-pin SCART connector. e Set the AV1 OUT switch to the appropriate position. See "AV1 INPUT/OUTPUT SIGNAL SELECTION FOR AV CONNECTION" below.
21-pin SCART connector RF Cable (provided)

TV Aerial Cable

21-pin SCART Cable (provided)
Mains outlet AV1 OUT switch
Mains power inlet AV1 IN/OUT Rear view
CONNECT RECORDER TO MAINS
Connect the provided mains power cord between the mains power inlet on the rear panel of the recorder and a mains outlet.
After you plug the mains power cord into a mains outlet, the Country Set display appears on the TV screen and/or on the recorder's front display panel when the button on the recorder/remote control is pressed for the first time to power on the recorder; go to page 6 to perform Auto Set Up.

ATTENTION:

Your TV must have a 21-pin AV input connector (SCART) for the connection to the recorder.
AV1 INPUT/OUTPUT SIGNAL SELECTION FOR AV CONNECTION
The AV1 IN/OUT connector accepts and delivers either a composite signal (regular video signal) or a Y/C signal (a signal in which the luminance and chrominance signals are separated). For input signal selection, select "VIDEO" (regular video signal) or "S-VIDEO" (Y/C signal) for "AV1 SELECT" setting ( pg. 54). For output signal selection, use AV1 OUT switch of the rear panel. If your TV's 21-pin AV input connector (SCART) is compatible only with the regular video signal, set this switch to COMP. If your TV's 21-pin AV input connector (SCART) is compatible with the Y/C signal, set this switch to Y/C. You can obtain highquality S-VHS pictures. (For connection, be sure to use a 21-pin SCART cable that is compatible with the Y/C signal.)

NOTES:

Set your TV to the VIDEO (or AV), Y/C, or RGB mode according to the type of your TV's SCART connector. For switching the TV's mode, refer to the instruction manual of your television. To obtain high-quality S-VHS pictures, you can also use the S-VIDEO CONNECTION described on page 5.

S-VIDEO Connection

Back of TV Aerial terminal S-VIDEO IN connector AUDIO IN connectors S-Video cable (provided)

Press % to move the highlight bar (pointer) to the language of your choice.
DANSK SUOMI NORSK SVENSKA NEDERLANDS CASTELLANO ITALIANO FRANCAIS DEUTSCH ENGLISH [5] = [MENU] : ENDE
(Ex.) DEUTSCH is selected

PERFORM AUTO SET UP

Press OK.
If you are referring to the front display panel Press % to select "Auto" and press OK or #.
Press % to move the highlight bar (pointer) to "AUTO SET" and press OK or #.

AUTO SET T-V LINK

[5] = [MENU] : EXIT
"Auto" blinks on the display panel; do NOT press any button on the recorder or remote control until the display panel shows clock time, "(CH) 1" or " : " as illustrated on page 9. If you are using the onscreen display, the AUTO SET screen will appear. As Auto Set Up progresses, the " " mark on the screen moves from left to right.

AUTO SET PLEASE WAIT

[MENU] : EXIT

Beginning

INITIAL SETTINGS (cont.)
When you connect the recorder and your TV via fully-wired 21-pin SCART cable ( pg. 4), you can set the recorder's tuner channels by downloading preset data from your TV instead of using the Auto Set Up function ( pg. 6). After downloading is completed, the recorder sets the clock and Guide Program number automatically. For details, refer to the instruction manual for your TV. Perform steps 1 to 3 of "Auto Set Up" on page 6 and 7 before continuing.

Preset Download

You can use this function only with the TV offering T-V Link, etc.*
* Compatible with TVs offering T-V Link, EasyLink, Megalogic, SMARTLINK, Q-Link, DATA LOGIC or NEXTVIEWLINK via fullywired 21-pin SCART cable. The degree of compatibility and available functions may differ by system.

PERFORM PRESET DOWNLOAD

If you are referring to the front display panel Press % to select "CH " and press OK or #.
Press % to move the highlight bar (pointer) to "T-V LINK" and press OK or #.
Preset position on the display panel increases from "CH1"; do NOT press any button on the recorder or remote control until the display panel shows clock time, "(CH) 1" or " : " as illustrated on page 9. If you are using the onscreen display, the T-V LINK screen will appear. Then the GUIDE PROG SET screen will appear during Guide Program number set. If you press any button on the recorder or remote control while downloading is in progress, it will be interrupted.

T-V LINK PLEASE WAIT

GUIDE PROG SET PLEASE WAIT
In the area where no TV station transmits a PDC signal, the recorder can perform neither auto clock set nor auto Guide Program number set. If there is a power cut, or if you press or MENU while downloading or set up is in progress, it will be interrupted; be sure to turn off the recorder power once and try again from the beginning. Auto clock may not function properly depending on the reception condition. When you perform T-V LINK function, be sure to use fullywired 21-pin SCART cable. On this recorder, the characters available for stations name (ID) are AZ, 09, , , + and (space). Some downloaded stations name may differ from those of your TV ( pg. 66).
Results of Auto Set Up/Preset Download appear on the front display panel
When both auto channel set and auto clock set have been completed successfully the correct current time will be displayed.
When auto channel set has been completed successfully but auto clock set has not, "1" (channel position) will be displayed.
When neither auto channel set nor auto clock set has been completed successfully, " : " will be displayed.
You can check if the Guide Program numbers have been set correctly when you perform the SHOWVIEW Timer Programming ( pg. 30); if the correct channel position number is displayed in step 3, this confirms that the Guide Program number for the SHOWVIEW number you enter in step 2 has been set correctly.
A If both auto channel set and auto clock set have been performed successfully: 1 Turn on the TV and select its AV mode, then make sure that all necessary stations have been stored in the recorder's
memory by using the TV PROG button(s). If station names (ID pg. 67) have also been stored in the recorder's memory, the station name will be displayed at the top left corner of the screen for about 5 seconds when the recorder is tuned to a different station. If you want to set the tuner manually such as to add or skip channels, to change channel positions, or to set or change station names, see pages 64 68.
B If auto channel set has succeeded but auto clock set has not: 1 Turn on the TV and select its AV mode, then make sure that all necessary stations have been stored in the recorder's
memory by using the TV PROG button(s). If station names (ID pg. 67) have also been stored in the recorder's memory, the station name will be displayed at the top left corner of the screen for about 5 seconds when the recorder is tuned to a different station. If you want to set the tuner manually such as to add or skip channels, to change channel positions, or to set or change station names, see pages 64 68. 2 Perform "Clock Set" on page 70.
C If both auto channel set and auto clock set have failed: 1 Make sure the TV aerial cable is connected properly to the recorder and turn off the recorder power once, then turn the

Normally it is recommendable to keep "DIGITAL 3R" set to "ON". Depending on the type of tape being used, picture quality may be better with "DIGITAL 3R" set to "OFF".

Repeat Playback

Your video recorder can automatically play back the whole tape 50 times repeatedly.

AUDIO2

FF PAUSE

Press PLAY.

ACTIVATE REPEAT PLAYBACK
Press PLAY and hold for over 5 seconds, then release. The Play indicator ( ) on the display panel blinks slowly. The tape plays 50 times automatically, and then stops.

REW STOP

Press STOP at any time to stop playback.
Pressing PLAY, REW, FF or PAUSE also stops Repeat Playback. For a cassette recorded in D-VHS mode, Repeat Playback is possible only if the cassette was recorded in STD mode.
Soundtrack Selection (S-VHS/VHS Mode)
Your video recorder is capable of recording three soundtracks (Hi-Fi L, Hi-Fi R and NORM) in the S-VHS/VHS mode and will play back the one you select.

During Playback

Pressing AUDIO on the remote control changes the soundtrack being played back as follows: TRACK Recorder's Front Panel + On-Screen Display L L R NORM + + NORM L NORM R NORM R USE For Hi-Fi stereo tapes For main audio of Bilingual tapes For sub audio of Bilingual tapes For audio-dubbed tapes For audio-dubbed tapes
" + " should normally be selected. In this mode, Hi-Fi stereo tapes are played back in stereo, and the normal audio track is played back automatically for tapes with only normal audio. For instructions on recording stereo and bilingual programmes, refer to page 27. "O.S.D." must be set to "ON" or the on-screen displays will not appear ( pg. 11).
Soundtrack Selection (D-VHS Mode)
Your video recorder is capable of recording two soundtracks (L and R) in the D-VHS mode and will play back the one you select.
Pressing AUDIO on the remote control changes the soundtrack being played back as follows: TRACK Recorder's Front Panel + On-Screen Display L L R R USE For stereo tapes For main audio of Bilingual tapes For sub audio of Bilingual tapes

D-VHS Recording

Basic Recording
TV signals being received by the recorders built-in tuner can be recorded onto a video tape. You can "capture" a TV programme using your video recorder. Turn on the TV and select the AV mode.
Insert a D-VHS cassette with the record safety tab intact. The recorder power comes on automatically and the counter is reset to 0:00:00. The STD (or LS3) indicator lights and the D-VHS recording mode is selected automatically.

SELECT S-VHS MODE

Move the highlight (pointer) to "S-VHS" by pressing %, then press OK or # to select "AUTO" or "OFF".

AUDIO 2

When playing back a tape that has been recorded in S-VHS, the S-VHS indicator lights on the display panel regardless of the "S-VHS" mode setting.
Receiving Stereo And Bilingual Programmes
Your recorder is equipped with a Sound-Multiplex decoder (A2) and a Digital stereo sound decoder (NICAM) making reception of stereo and bilingual broadcasts possible. When the channel is changed, the type of broadcast being received will be displayed on the TV screen for a few seconds. Type of Broadcast Being Received A2 Stereo A2 Bilingual Regular Monaural NICAM Stereo NICAM Bilingual NICAM Monaural On-screen Display ST BIL (none) ST NICAM BIL NICAM NICAM
To Record NICAM Stereo And Bilingual Programmes (Only For The Users In Eastern Europe)
S-VHS/VHS mode The NICAM audio programme will be recorded on the Hi-Fi audio track, and the Standard audio programme on the normal audio track. D-VHS mode (See "Audio Rec Mode Setting" below.)
If the quality of stereo sound being received is poor, the broadcast will be received in monaural with better quality. Before playing back a programme recorded in stereo, or a bilingual programme, refer to "Soundtrack Selection" ( pg. 21).
To listen to a stereo programme, press AUDIO until " " and " " appear on the front display panel or "L R" appears on the screen. To listen to a bilingual programme, press AUDIO until " " or " " appears on the front display panel, or "L " or " R" appears on the screen (as required). S-VHS/VHS mode To listen to the Standard (regular monaural) audio while receiving a NICAM broadcast, press AUDIO until "NORM" appears on the front display panel or on the screen.
Audio Rec Mode Setting (D-VHS Mode Only)
1 Press MENU to access the Main Menu screen. 2 Move the highlight bar (pointer) to "MODE SET" by pressing %, then press OK or #. 3 Move the highlight bar (pointer) to "AUDIO REC" by pressing %, then press OK or # to select "NICAM" or

"MONO".

"O.S.D." must be set to "ON" or the on-screen displays will not appear ( pg. 11).
To Record Stereo And Bilingual Programmes (A2)
S-VHS/VHS mode Stereo programmes are automatically recorded in stereo on the Hi-Fi audio track (with the normal audio track recording mixed L and R channel sound). Bilingual programmes are automatically recorded in bilingual on the Hi-Fi audio track. The main soundtrack will be recorded on the normal audio track. D-VHS mode Stereo programmes are automatically recorded in stereo on the audio track. Bilingual programmes are automatically recorded in bilingual on the audio track.
4 Press MENU to return to the normal screen.

Auto Timer

When the Auto Timer is set to ON the timer is automatically engaged when the recorder power is turned off and automatically disengaged when the recorder is powered back on.
Press % to move the highlight bar (pointer) to "AUTO TIMER", then press OK or # to select either "ON" or "OFF".
MODE SET B. E. S. T. ON PICTURE CONTROL AUTO AUTO TIMER ON O. S. D. ON DIRECT REC ON AUTO SP=LP TIMER OFF DIGITAL 3R ON NEXT PAGE [5] = [MENU] : EXIT
For safety, when Auto timer is set to "OFF", all other recorder functions are disabled while the Timer mode is engaged. To disengage the timer, press (or TIMER).
Automatic Satellite Programme Recording

C LI N K RE

This facility allows you record automatically a satellite programme which is timer-programmed on your external satellite tuner. Connect a satellite tuner to the recorder's AV2 IN/DECODER connector ( pg. 57, 58) and programme the timer on the satellite tuner; the recorder starts recording when the signals input from the satellite tuner to the AV2 IN/DECODER connector, and when there is no input signals the recorder stops recording and the power shuts off. Before performing the following steps: Make sure the satellite tuner is connected to the recorder's AV2 IN/DECODER connector. ( pg. 57, 58) Programme the timer on the satellite tuner. Insert a cassette with the safety tab in place. Set the appropriate recording mode (D-VHS, S-VHS or VHS) ( pg. 22, 23).
Set "AV2 SELECT" to "AV2" ( pg. 54).

SELECT AV2 SELECT MODE

When you select "SAT", refer to "IMPORTANT" on page 57.

REC LINK indicator

Be sure not to turn on the satellite tuner before the programme is executed; otherwise, the recorder will start recording when the satellite tuner's power is turned on. If you have connected another appliance other than a satellite tuner to the AV2 IN/DECODER connector, be sure not to engage the Auto Satellite Prog Rec mode; otherwise, the recorder will start recording when the connected appliance's power is turned on. Auto Satellite Prog recording and timer-recording cannot be done at the same time.
ENGAGE AUTO SATELLITE PROG REC MODE
Press and hold REC LINK on the recorder for about 2 seconds. The REC LINK indicator lights up and the recorder turns off automatically. To disengage the Auto Satellite Prog Rec mode, press REC LINK. The REC LINK indicator goes off. If the recorder's power is off, it is not possible to engage the Auto Satellite Prog Rec mode.

Auto Satellite Prog recording is possible with the AV2 IN/DECODER connector only. ( pg. 57, 58) For timer programming of the satellite tuner, refer to the instruction manual of the satellite tuner. Auto Satellite Prog recording is not possible if your satellite tuner does not have a timer. The REC LINK indicator blinks while Auto Satellite Prog recording is in progress. Pressing the recorder's button while Auto Satellite Prog recording is in progress turns off the recorder's power and disengage the Auto Satellite Prog Rec mode. If there are more than one satellite programme you wish to record with Auto Satellite Prog Recording, it is not possible to set the different tape speed per each; the tape speed selected in step 2 will be applicable to all the programmes for Auto Satellite Prog recording. The B.E.S.T. system ( pg. 28) does not work while Auto Satellite Prog recording is in progress. Just Clock ( pg. 70) does not work when the Auto Satellite Prog Rec mode is engaged. Depending on the type of satellite tuner, the recorder may not record a slight portion of the beginning of the programme or may record slightly longer than the actual length of the programme. If you engage the Auto Satellite Prog Recording mode when the satellite tuner's power is on, the recorder will not start Auto Satellite Prog recording even though the REC LINK indicator blinks. When the satellite tuner shuts off once and is turned back on again, the recorder starts recording. When the Auto Satellite Prog Rec mode is engaged or the recorder's power is turned off after Auto Satellite Prog recording is finished, the recorder will not enter the Timer mode even though "AUTO TIMER" is set to "ON". You can also record a programme from your cable system in the same way if the system has a timer. When you press and hold the recorder's REC LINK button to engage the Auto Satellite Prog Rec mode, if the REC LINK indicator does not light but instead blinks quickly even though your satellite tuner's power is off, Auto Satellite Prog Recording will not work properly with that satellite tuner*. If this is the case, perform "Express Timer Programming" ( pg. 32) to timer-record a satellite programme.

NUMBER "0"

SET RECORDING MODE
Set the appropriate recording mode (D-VHS, S-VHS or VHS) ( pg. 22, 23).

SET RECORDERS INPUT MODE

Press NUMBER key "0" and/or TV PROG to select "F-1" for the AUDIO/VIDEO input connectors, "S-2" for the AUDIO/S-VIDEO input connectors, or "I-1" for the DV input connectors, depending on the connectors being used.
SET EDIT MODE (S-VHS/VHS MODE ONLY)
See "Picture Control" on page 49.

START CAMCORDER

Engage its Play mode.
All necessary cables can be obtained from your dealer. You can also use another video recorder as the player instead of a camcorder. When you select EDIT to dub tapes in step 4, be sure to select AUTO (or NORM when B.E.S.T. is set to OFF) after you finish dubbing the tapes. When you are editing through the DV IN connector, the recorder will stop if the player begins playing a blank portion of tape or the signal is interrupted.

START RECORDER

Engage its Record mode.
A If the camcorder has no S-VIDEO output connector. AUDIO input Recorder AUDIO input
B If the camcorder has an S-VIDEO output connector. Recorder
To rear panel R.PAUSE connector VIDEO input Audio Cable (not provided) Video Cable (not provided) Player Camcorder VIDEO OUT S-VIDEO input
To rear panel R.PAUSE connector
Audio Cable (not provided) S-Video Cable (provided) S-VIDEO OUT Mini-Plug Cable (not provided) (JVC camcorder only)
Mini-Plug Cable (not provided) (JVC camcorder only)

Player Camcorder

AUDIO OUT EDIT
C If the camcorder has a DV OUT connector. Recorder

DV Sound Setting

Up to four audio signal channels can be input from a DV camcorder through the DV IN connector (i.Link). This recorder allows you to select and record two of these channels. This setting is used to specify which two channels to record.
1 Press MENU to access the Main Menu screen. 2 Move the highlight bar (pointer) to "MODE SET" by pressing %, then press OK or #. 3 Move the highlight bar (pointer) to "DV SOUND" by pressing %, then press OK or # to select "SOUND1",

PREVIOUS PAGE POWER SAVE OFF S-VHS AUTO AV1 SELECT VIDEO AV2 SELECT AV2 COLOUR SYSTEM PAL/NTSC DV SOUND SOUND1 AUDIO REC NICAM [5] = [MENU] : EXIT

AV2 SELECT Setting

Set "AV2 SELECT" to the appropriate mode depending on the type of unit connected to the rear panel AV2 IN/DECODER connector of this recorder. 1 Press MENU to access the Main Menu screen. MODE SET 2 Press % to move the highlight bar (pointer) to "MODE SET", then press OK or #. B. E. S. T. ON 3 Press % to move the highlight bar (pointer) to "AV2 SELECT". PICTURE CONTROL AUTO AUTO TIMER OFF 4 Press OK or # to select "AV2", "DECODER" or "SAT". O. S. D. ON aAV2 : To use this recorder as the recording deck with the player connected to the DIRECT REC ON AUTO SP=LP TIMER OFF AV2 IN/DECODER connector, or to use the satellite tuner connected to the DIGITAL 3R ON AV2 IN/DECODER connector. NEXT PAGE bDECODER : To use a decoder connected to the AV2 IN/DECODER connector. [5] = [MENU] : EXIT cSAT : To view a satellite programme with the TV set while the recorder is in Timer mode, in Stop mode, recording or turned off. ( pg. 57) 5 Press MENU to return to normal screen. If you have a decoder connected to the AV2 IN/DECODER connector, be sure to set "AV2 SELECT" back to "DECODER" after editing. If you're not connecting a decoder to the AV2 IN/DECODER connector, leave "AV2 SELECT" set to "AV2". The default setting is "AV2"; if the recorder's memory backup has expired due to a power cut or because the AC was removed from the recorder, "AV2" will be automatically selected when the power is restored to the recorder. If you are using a decoder, be sure to set "AV2 SELECT" back to "DECODER". If the AV1 OUT switch on the rear panel is set to Y/C, it is impossible to set "AV2 SELECT" to "DECODER".

Synchro Editing

The Synchro Editing function synchronizes the start of the playback and recording operations when starting an edit operation using a camcorder with LANC connector and your video recorder.
Connect your recorder to camcorder ( pg. 50), and connect your recorder's SYNCHRO EDIT connector to the camcorder's LANC connector.
Set the appropriate recording mode (S-VHS or VHS) ( pg. 23).
SET RECORDER'S INPUT MODE
Set the appropriate input mode, depending on the connectors being used in step 1.

SET EDIT MODE

LOCATE START POINT
Start playback of the tape in the camcorder, and pause playback when you find the point where you want to start editing. Press and hold PAUSE and press RECORD on your recorder so that the recorder enters the RecordPause Mode.

SYSTEM CONNECTIONS (cont.)
Set "AV2 SELECT" to "AV2". ( pg. 54) You can use Automatic Satellite Program Recording function ( pg. 37) with this connection. To record a programme via the satellite tuner, select L-2 mode by pressing NUMBER keys and/or TV PROG so that "L-2" appears on the display panel. To receive a scrambled broadcast, descramble the signal with the satellite tuner. For details, refer to the instruction manual for the satellite tuner and decoder.
21-pin AV input connectors (SCART) Back of TV TV Aerial cable

Aerial connector

Decoder
Back of Recorder Mains outlet

Basic Connections

This is an example of basic connection if you have both satellite tuner and decoder.
Connect the decoder to the satellite tuner's connector and AV1 IN/OUT connector to the satellite tuner's connector. Then connect the satellite tuner and TV.
To record a satellite programme which delivers the Y/C signal, set "AV2 SELECT" to "AV2" and "AV1 SELECT" to "S-VIDEO". You can obtain high-quality S-VHS picture. If you connect your recorder's S OUT and AUDIO OUT connectors to the TV, you can enjoy high-quality S-VHS pictures. Automatic Satellite Program Recording function is not possible with this connection. To record a programme via the satellite tuner, select L-1 mode by pressing NUMBER keys and/or TV PROG so that "L-1" appears on the display panel. To receive a scrambled broadcast, descramble the signal with the satellite tuner. For details, refer to the instruction manual for the satellite tuner and decoder.
Outdoor Unit Satellite Cable
Aerial Aerial connector TV Aerial cable Back of TV

Mains outlet

The AV2 IN/DECODER connector can be used as an input terminal for an external decoder (descrambler). Simply connect a decoder and you can enjoy the variety of programming that is available through scrambled channels.
Connecting/ Using A Decoder
TV Receiver RF Cable (provided)
TV Aerial Cable AV1 IN/OUT
Set "AV2 SELECT" to "DECODER". ( pg. 54)

SELECT INPUT MODE

CONNECT DECODER
Connect your recorder's AV2 IN/DECODER connector to the decoder's Euroconnector using a 21-pin SCART cable.

SET TUNER

Perform the procedure as described in "When Receiving A Scrambled Broadcast" on page 65.

AV2 IN/DECODER

Connecting/ Using A Stereo System

FM Tuner CD Player

These instructions enable you to connect your video recorder to your Hi-Fi stereo system (if you have one) and listen to the soundtrack through the stereo.

If you are using the AV or S-VIDEO connection, set the TV to its AV mode. Try manual tracking. ( pg. 17) Consult your JVC dealer.
Automatic tracking is engaged.
Engage and adjust tracking manually ( pg. 17), or set the soundtrack to "NORM" ( pg. 21).
1. Recording cannot be started.
There is no cassette loaded, or the cassette loaded has had its Record Safety tab removed. "I-1", "L-1", "L-2", "F-1", "S-1" or "S-2" has been selected as the input mode. The source (another video recorder, camcorder) has not been properly connected. All necessary power switches have not been turned on. The input mode is not correct. The camcorder has not been properly connected. The input mode is not correct.
Insert a cassette, or using adhesive tape, reseal the slot where the tab was removed. Set to the desired channel. Confirm that the source is properly connected. Confirm that all units power switches are turned on. Set the input mode to "I-1", "L-1", "L-2", "F-1", "S-1" or "S-2". Confirm that the camcorder is properly connected. Set the input mode to "I-1", "L-1", "L-2", "F-1", "S-1" or "S-2".
2. TV broadcasts cannot be recorded. 3. Tape-to-tape editing is not possible.
4. Camcorder recording is not possible.
1. Timer recording wont work.
The clock and/or the timer have been set incorrectly. The timer is not engaged. Timer recording is in progress.
Re-perform the clock and/or timer settings. Press and confirm that "" is displayed on the display panel. Timer programming cant be performed while a timer recording is in progress. Wait until it finishes. Load a cassette with the Record Safety tab intact, or cover the hole using adhesive tape. Remove the cassette and cover the hole with adhesive tape, or insert a cassette with the Record Safety tab intact. Check the programmed data and reprogram as necessary, then press again. The programme may not have been recorded in its entirety. Next time make sure you have enough time on the tape to record the entire programme. When the front panel is closed and the cassette cannot be ejected fully, push the cassette into the recorder, then press EJECT to remove the cassette. Refer to "Guide Program Number Set" and re-perform the procedure ( pg. 69).
2. Timer programming is not possible.
3. "" and "]" on the display panel wont stop blinking. 4. The cassette is automatically ejected, and "" and "]" on the display panel wont stop blinking. 5. "" blinks for 10 seconds and the Timer mode is disengaged.

(Ex.) FRANCAIS is selected for BELGIUM Language Code
ENGLISH DEUTSCH FRANCAIS ITALIANO CASTELLANO : : : : : 05 NEDERLANDS SVENSKA NORSK SUOMI DANSK : : : : : 10

Language Code

Press OK (remote). "Auto" appears on the display panel.
Press OK (remote) to start Auto Set Up. Do NOT press any button on the video recorder or remote control during "Auto" is blinking.
If "(CH) 1" or " : " appears on the display panel, see page 9 of the instructions.
* Once you have performed Auto Set Up, even if the recorder's memory backup has expired, the recorder will not perform Auto Set Up again. Perform tuner setting ( pg. 62) and/or clock setting ( pg. 70) as required.
FOR MORE INFORMATION SEE INSTRUCTIONS/P6
VICTOR COMPANY OF JAPAN, LIMITED
COPYRIGHT 1999 VICTOR COMPANY OF JAPAN, LTD.
Printed in Japan 1099MNV ID VP

Comparative Hepatology

Research

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A remission spectroscopy system for in vivo monitoring of hemoglobin oxygen saturation in murine hepatic sinusoids, in early systemic inflammation
Christian Wunder*1, Robert W Brock3, Alfons Krug2, Norbert Roewer1 and Otto Eichelbrnner1
Address: 1Klinik und Poliklinik fr Ansthesiologie, Julius-Maximilians-Universitt Wrzburg, Zentrum fr Operative Medizin, Oberdrrbacher Strasse 6, 97080 Wrzburg, Germany, 2LEA Medizintechnik GmbH, 35394 Giessen, Germany and 3Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, 72205-7199 Little Rock, USA Email: Christian Wunder* - christian.wunder@mail.uni-wuerzburg.de; Robert W Brock - BrockRobertW@uams.edu; Alfons Krug - krug@lea.de; Norbert Roewer - dir.anaesth@klinik.uni-wuerzburg.de; Otto Eichelbrnner - oeichelbroenner@anaesthesie.uni-wuerzburg.de * Corresponding author
Published: 12 January 2005 Comparative Hepatology 2005, 4:1 doi:10.1186/1476-5926-4-1
Received: 20 October 2004 Accepted: 12 January 2005
This article is available from: http://www.comparative-hepatology.com/content/4/1/Wunder et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: During the early stages of systemic inflammation, the liver integrity is compromised by microcirculatory disturbances and subsequent hepatocellular injury. Little is known about the relationship between the hemoglobin oxygen saturation (HbsO2) in sinusoids and the hepatocellular mitochondrial redox state, in early systemic inflammation. In a murine model of early systemic inflammation, we have explored the association between the sinusoidal HbsO2 detected with a remission spectroscopy system and 1.) the NAD(P)H autofluorescence (an indicator of the intracellular mitochondrial redox state) and 2.) the markers of hepatocellular injury. Results: Animals submitted to 1 hour bilateral hindlimb ischemia (I) and 3 hours of reperfusion (R) (3.0 h I/R) exhibited lower HbsO2 values when compared with sham. Six hours I/R (1 hour bilateral hindlimb ischemia and 6 hours of reperfusion) and the continuous infusion of endothelin-1 (ET-1) further aggravated the hypoxia in HbsO2. The detected NAD(P)H autofluorescence correlated with the detected HbsO2 values and showed the same developing. Three hours I/R resulted in elevated NAD(P)H autofluorescence compared with sham animals. Animals after 6.0 h I/R and continuous infusion of ET-1 revealed higher NAD(P)H autofluorescence compared with 3.0 h I/R animals. Overall the analysed HbsO2 values correlated with all markers of hepatocellular injury. Conclusion: During the early stages of systemic inflammation, there is a significant decrease in hepatic sinusoidal HbsO2. In parallel, we detected an increasing NAD(P)H autofluorescence representing an intracellular inadequate oxygen supply. Both changes are accompanied by increasing markers of liver cell injury. Therefore, remission spectroscopy in combination with NAD(P)H autofluorescence provides information on the oxygen distribution, the metabolic state and the mitochondrial redox potential, within the mouse liver.

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(page number not for citation purposes)
Comparative Hepatology 2005, 4:1
http://www.comparative-hepatology.com/content/4/1/1

Background

Hepatic microcirculatory failure is a major prerequisite for the development of hepatocellular dysfunction in a number of conditions like trauma/hemorrhage, liver transplantation and systemic inflammation. In various inflammatory states, the degree of lethal hepatocyte necrosis can be predicted from the extent of hepatic microcirculatory failure [1], possibly via alterations in the mitochondrial redox state of the liver [2,3]. Previously, our group has shown that the development of systemic inflammation was associated with a disturbance of the hepatic microcirculation, and a subsequent increase in hepatocellular damage [4,5]. The causal mechanisms are not completely understood, but accumulating evidence suggests a dysregulation of stress-inducible vasoactive mediators like endothelins, nitric oxide synthase or heme oxygenase [6]. Moreover, modifications in effector cell function may also alter the response to those mediators [7]. Hepatic microcirculatory failures during various stresses are typically characterized by alterations in the distribution of perfusion, thereby resulting in a disparity between oxygen supply and demand. This impaired nutritive blood flow, together with reduced oxygen availability, decreases cellular high-energy phosphates leading to an early hepatocellular injury and dysfunction. Studies of tissue oxygenation focusing on the relationship between microcirculatory disturbances and oxygen transport dynamics may help to better elucidate the pathophysiological mechanisms involved. Several methods have been reported in the past couple of years directly quantifying the oxygen distribution in tissues; however, their applicability in tissues, especially in small rodents like mice, is limited due to technical reasons. For instance, microelectrodes measure tissue pO2 at specific points; but the technique is invasive and consumes oxygen. Electron paramagnetic resonance oximetry techniques or nuclear MRI approaches allow the detection of changes in tissue pO2; however, their resolution is too low [8]. A fluorescent membrane, developed by Itoh et al. [9] on the basis of an oxygen-quenched fluorescent dye allows the in vivo visualization of the tissue pO2. This techTable 1: Arterial blood gases.

nique allows the visualization of oxygen distribution on tissue surfaces, but this method comprised some technical limitations. The oxygen-sensitive membrane has to be used under gastight and watertight conditions during microscopy and the fluorescent membrane shows a photobleaching effect. Paxian et al. [10] recently demonstrated that the intravenous infusion of a special oxygen quenching dye allowed the visualization of the oxygen distribution on the liver surface using intravital videomicroscopy. The fluorescence of the dye was directly dependent on the tissue pO2. A disadvantage of this method, especially when used in small rodents like mice, is that it requires changing the continuous intravenous infusion rates of the dye to provide stable plasma concentrations. With mice (increasingly used as laboratory animals) there is a growing need for a method able to reliably detect tissue oxygenation or, at least, hemoglobin oxygen saturation (HbsO2) in capillaries of small animals. The aim of the present study was to investigate whether the utility of a new and simple remission spectroscopy system allows reliable in vivo detection of liver sinusoidal HbsO2. In a mouse model of early systemic inflammation, we examined whether the detected changes in hepatic HbsO2 correlated with the established method of NAD(P)H autofluorescence and hepatocellular injury.

Results

Macrohemodynamics Consistent with previous reports [4,11], mean arterial pressure (MAP) was significantly lower in animals after ischemia (I) and reperfusion (R) (3.0 h I/R and 6.0 h I/R) compared to sham animals, but remained normotensive (> 80 mmHg) throughout the study. MAP did not differ between the I/R groups. Central venous pressure was not different (data not shown). Blood gas analysis The measurement of arterial blood gases carried out after the microscopy procedure showed normal oxygenation, a moderate acidosis, and adequate pCO2 for all groups
pO2 (mmHg) Sham 3.0 h I/R 6.0 h I/R 6.0 h I/R+endothelin-(46) 123 (49) 116 (38) 119 (46)
pH 7.29 (0.13) 7.27 (0.15) 7.26 (0.17) 7.26 (0.13)
pCO2 (mmHg) 35.8 (11.3) 36.7 (10.8) 36.8 (12.4) 36.9 (12.9)
Data expressed as Mean (SD); n = 7 for each group

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(Table 1).
Hepatic sinusoidal hemoglobin oxygen saturation (HbsO2) Hepatic sinusoidal HbsO2 of the different groups are shown in Figure 1. Animals treated with 3.0 h I/R have significant lower hepatic HbsO2 values (56.2 (13.1)) when compared with sham (68.4 (14.1); p < 0.01). No statistically significant differences were observed between 3.0 h I/R and 6.0 h I/R treated animals. However, an obvious shift of hepatic HbsO2 towards a lower oxygenation was observed when compared with 3.0 h I/R treated animals. Animals treated with 6.0 h I/R and a continuous infusion of endothelin-1 (ET-1) showed significant reduced HbsO2 values (44.8 (14.7)) when compared with 3.0 h I/R treated animals (56.2 (13.2); p < 0.006). More than half of the measured data from these animals revealed HbsO2 values lower than 50%. There was no apparent difference in the local tissue hemoglobin (Hb) content detected (data not shown). Hepatic tissue redox status Animals subjected to 3.0 h I/R revealed significantly higher NAD(P)H autofluorescence (141.6 (12.8)); therefore, a significant decline in hepatic tissue oxygenation was observed when compared with sham (100.0 (6.7)) (Figure 2). Three hours I/R treated animals failed to show a significant difference in NAD(P)H autofluorescence when compared with the 6.0 h I/R treated animals. Animals treated with 6.0 h I/R and a continuous infusion of ET-1 demonstrated significantly higher NAD(P)H autofluorescence (161.1 (13.8)) when compared to the 3.0 h I/R treated animals (141.6 (12.8)). There was a highly significant correlation found between NAD(P)H autofluorescence and hepatic HbsO2 detected in the same animal (p < 0.005; r2 = 0.94), as depicted in Figure 3. Hepatic tissue injury Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) levels are summarized in Table 2. When compared with sham animals, mice treated with 3.0 h I/R exhibited significantly higher levels of ALT and AST. No significant changes between 3.0 h I/R and 6.0 h I/R animals were detectable. When compared with 3.0 h I/R, mice treated with 6.0 h I/R and a continuous infusion of ET-1 showed significant higher ALT and AST levels. The results of labelling lethally injured hepatocytes with propidium iodide (PI) are shown in Figure 4. The 3.0 h I/R treated animals exhibited a significantly increase in lethally injured hepatocytes (120.4 (44.0)) compared with sham (25.7 (17.9)), whereas the 6.0 h I/R group had a significant higher number of dead hepatocytes (260.1 (52.7)) than the 3.0 h I/R treated animals. The treatment of 6.0 h I/R animals with a continuous ET-1 infusion further elevated the degree of lethally injured hepatocytes (361.8 (56.0)) when compared to the 6.0 h I/R treated

animals. Regression analysis between lethally injured hepatocytes and hepatic HbsO2 revealed a significant correlation (p < 0.001; r2 = 0.86), as shown in Figure 5.

Discussion

In the present study, we demonstrate the utility of a remission spectroscopy system for the in vivo measurement of murine hepatic sinusoidal HbsO2 that showed a significant correlation with the established method of NAD(P)H autofluorescence, as well as with the extent of hepatic tissue injury. Oximetry relies on the detection of the spectral properties of oxygenated and reduced Hb. In vitro bench analysis capabilities have spurred the desire to accomplish accurate in vivo measurement through various techniques. The 1930's and 1940's were a particularly active period for oximetry advances culminating in the development of pulse oximeters in the 1970's [12]. Remission spectroscopy is based on the same principles of those oximeters, namely because they rely on the emission of white light and measure the total intensity of the backscattered light returned from the tissue. The intensity of the backscattered light is dependant on the amount and absorbance of the Hb in the tissue under observation. Oxygenated Hb has a different absorbance from that of deoxygenated Hb. The analysis of the backscattered light spectrum allows the determination of the HbsO2 in the tissue. Previously, it has been shown that bilateral hindlimb I/R results in the deterioration of liver microcirculation [13]. Since the hepatic Hb content was not found to be different between groups in this study, the differences in the backscattered light spectra only represent differences in the HbsO2. In the past, we have shown that bilateral hindlimb I/R results in a systemic inflammation with hepatic microcirculatory disturbances, in terms of reduced sinusoidal diameters and sinusoidal volumetric blood flow accompanied by elevated levels of sinusoidal leukocytes [4,5]. These disturbances may result in an imbalance between oxygen supply and oxygen demand. Since the spectra, extinction coefficient, and quantum yield of NADH and NADPH are the same [14,15], they are designated together as NAD(P)H this naturally occurring fluorophore transfers electrons to oxygen by means of an electron transport chain located at the inner membrane of mitochondria [16]. Under hypoxic conditions, with no oxygen available to accept electrons from cytochrome a, intracellular NAD(P)H accumulates. Unlike the oxidized form NAD+, NAD(P)H is highly fluorescent [17]. Therefore, we compared the changes in NAD(P)H autofluorescence, which reflect the extent of tissue hypoxia, with that of hepatic HbsO2 obtained by the remission spectroscopy system under pathophysiological conditions. Whether induced by I/R or by the combination of I/R and infusion

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3.0 h I/R

Frequency

< -< -< -< -< -< -< -< -< -< -< 90 >9 0

Hepatic HbsO2

6.0 h I/R
Sinusoidal haemoglobin oxygen saturation (HbsO2) Figure 1 Sinusoidal haemoglobin oxygen saturation (HbsO2). At least 35 different observation points of the left liver lobe per animal were examined. The frequency distributions of all examined HbsO2 values per group are shown.

6.0 h I/R + endothelin-1

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NAD(P)H fluorescence (aU)
am Sh 3. 0 hI /R 6.0 /R hI 6. I 0h T-1 +E /R
of ET-1, both analytical methods showed a decrease in hepatic oxygen supply, either as an elevation in NAD(P)H autofluorescence or as a diminution in hepatic HbsO2. The significant correlation between remission spectroscopy and NAD(P)H fluorescence indicates that after 3.0 h I/R, 6.0 h I/R and 6.0 h I/R+ET-1, hepatic oxygen supply was compromised. This is further emphasized by the statistical relationship found between hepatic HbsO2 and the extent of subsequent hepatocyte death. Both remission spectroscopy and NAD(P)H autofluorescence provide information on the metabolic state of the murine liver. Remission spectroscopy is directly dependent on the HbsO2 in the sinusoids, whereas NAD(P)H autofluorescence depends upon the mitochondrial redox state and the activity of the mitochondrial electron transport chain. It was previously proposed that during systemic inflammation the NADH/NAD+ redox potential may increase, and oxygen utilization may be altered [18]. The present study demonstrates a concomitant change in NAD(P)H autofluorescence and hepatic HbsO2. Obviously, the observed hypoxia did not occur through altered oxygen utilization, but rather through a reduced oxygen supply induced by sinusoidal microcirculatory disturbances. This corroborates our previous contention that the simultaneous use of remission spectroscopy, and that of NAD(P)H autofluorescence, provides additional information regarding the underlying pathophysiological mechanisms. That technical approach allows the correlation between disturbances in oxygen supply and those of oxygen utilization.

Figure tissue redox status Hepatic 2 Hepatic tissue redox status. NAD(P)H autofluorescence, as a marker of the intracellular mitochondrial redox state, was examined using fluorescence intravital videomicroscopy with a filter set consisting of a 365 nm excitation and a 397 nm emission bandpass filter. The complete left liver lobe was systematically scanned and at least 15 different fields of view have been analysed. Fluorescence was densitometrically assessed and expressed as average intensity/liver acinus. * p < 0.001 vs. sham; # p < 0.01 vs. 3.0 h I/R; Data expressed as Mean + 2SD; n = 7 for each group.
Sinusoidal hemoglobin O2 saturation (%)
y = 116.05 - 0.44x 80 r2 = 0.94 p < 0.005 70

Conclusions

There is a significant reduction in hepatic sinusoidal HbsO2 during the early stages of systemic inflammation. In parallel, we detected an increasing NAD(P)H autofluorescence representing an intracellular inadequate oxygen supply. Both changes are accompanied by increasing markers of liver cell injury. Future therapeutic interventions should focus on the amelioration of sinusoidal HbsO2 followed by an improvement in mitochondrial redox state. Remission spectroscopy represents a simple and reliable method for hepatic sinusoidal HbsO2 determination in small rodents. In combination with NAD(P)H autofluorescence, it provides information on the oxygen distribution, the metabolic state and the mitochondrial redox potential within the hepatic tissue.
180 NADH fluorescence (aU)
tion (HbsO between sinusoidal hemoglobin oxygen saturaCorrelation2) and tissue redox status Figure 3 Correlation between sinusoidal hemoglobin oxygen saturation (HbsO2) and tissue redox status. The mean HbsO2 values significantly correlated with the corresponding NAD(P)H autofluorescence (p < 0.005; r2 = 0.94). Data derived from 32 animals.

Methods

Animals Male C57/BL6 mice (eight to ten weeks old, weighing 23.7 (11.1) g) were used for all experiments. The experimental protocols were in compliance with the guidelines of the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal

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Table 2: Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).
Sham ALT (U/L) AST (U/L) 50.2 (16.6) 177 (34)
3.0 h I/R 197.0 (40.4) * 1825 (410) *#
6.0 h I/R 226.2 (38.5) * 2551 (616) *
6.0 h I/R+endothelin-1 261.6 (37.8) *## 2856 (320) *##
Data expressed as Mean (SD); n = 7 for each group; * p < 0.001 vs. sham; # p < 0.02 vs. 6.0 h I/R; ## p < 0.01 vs. 3.0 h I/R.

y = 71.037- 0.0729 x 80 r2 = 0.867 p < 0.001 70
Lethal hepatocyte injury (PI-labeled nuclei / 10-1mm3)
0 Sham 3.0 h I/R 6.0 h I/R 6.0 h I/R+ET-1
500 Lethal hepatocyte injury (PI-labeled nuclei / 10-1mm3)
Figure tissue injury Hepatic 4 Hepatic tissue injury. Nuclei of lethaly injured hepatocytes were labelled in vivo with propidium iodide (PI). PI-labelled nuclei were quantified using fluorescence intravital videomicroscopy with a 510 to 560 nm excitation and an emission barrier filter greater than 590 nm. PI-labelled hepatocytes were expressed as number of cells/10-1mm3. * p < 0.001 vs. sham; # p < 0.001 vs. 3.0 h I/R; ## p < 0.01 vs. 6.0 h I/R; Data expressed as Mean + 2SD; n = 7 for each group.
tion (HbsO between sinusoidal hemoglobin Correlation2) and lethal hepatocyte injury oxygen saturaFigure 5 Correlation between sinusoidal hemoglobin oxygen saturation (HbsO2) and lethal hepatocyte injury. There is a significant correlation between the mean HbsO2 values and the corresponding amount of PI-labelled nuclei (p < 0.001; r2 = 0.87). Data derived from 32 animals.
Resources, National Research Council as well as those of Germany. Animals were maintained under controlled conditions (22C, 55% humidity and 12-hour day/night cycle) with free access to tap water and a standard laboratory chow.
Experimental protocol Mice (n = 7, for each group) were randomly assigned to either a Sham or a hindlimb ischemia/reperfusion (I/R) group. Animals of the I/R groups were treated with 60 minutes bilateral hindlimb ischemia induced by tightening a tourniquet above the greater trochanter of each leg while under anaesthesia. Sham animals were not subjected to ischemia, but remained anaesthetized for the same period of time. Tourniquets were removed just prior to recovery from anaesthesia. The animals were awake during the 3 hours (3.0 h I/R) or the 6 hours (6.0 h I/R) reperfusion periods, and re-anaesthetized for the intravital microscopy procedure.
To further induce liver microcirculatory disturbances and contribute towards a reduction in liver oxygen supply 6.0 h I/R, mice were further randomized to a group treated with a continuous infusion of ET-1 (70 pmol/min., i.v.) starting 15 minutes prior to microscopy. This dose of ET1 was chosen because it produced alteration in the oxygen distribution, along with derangements in the hepatic tissue perfusion [19].

Surgical procedure Animals received anaesthesia, by inhalation, for all procedures. As previously described [20], anaesthesia was performed using isoflurane (Forene, Abbott, Wiesbaden, Germany) in spontaneously breathing animals. The left carotid artery and the left jugular vein were cannulated under sterile conditions. The carotid artery cannula was used for the continuous measurement of systemic arterial blood pressure and heart rate, while central venous

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pressure was assessed via the jugular vein cannula. Throughout the experiment, normal saline was administered at a rate of 0.4 ml/hr to maintain normal mean arterial pressure. As formerly described [4], and for the realization of the intravital microscopy procedure in anaesthetized animals, a transverse subcostal incision was performed. Briefly, the ligament attachments from the liver to the diaphragm and to the abdominal wall were carefully released. For the evaluation of the hepatic microcirculation by intravital fluorescence microscopy, the animals were positioned on left lateral decubitus and the left liver lobe was exteriorized onto an adjustable stage. The liver surface was covered with a thin transparent film to avoid tissue drying and exposure to ambient oxygen. For equilibrium purposes, a pause of 10 minutes was allowed before data from microscopy and remission spectroscopy was collected. After microscopy, animals were killed by exsanguination, via the insertion of a cannula in the left femoral artery for the collection of arterial blood samples or via cardiac puncture.
Intravital microscopy Details of this technique have been described elsewhere [4,21]. For observations of the liver microcirculation, we used a modified inverted Zeiss microscope (Axiovert 200, Carl Zeiss, Gttingen, Germany) equipped with different lenses (Achroplan 10 NA 0.25 / 20 NA 0.4 / 40 NA 0.6). The image was captured using a 2/3" charge-coupled device video camera (CV-M 300, Jai Corp., Kanagawa, Japan) and digitally recorded (JVC HM-DR10000EU DVHS recorder) for off-line analysis. As previously described [22], NAD(P)H autofluorescence, as a marker of the mitochondrial redox state, was assessed using the 10x objective lens. The liver was examined using a filter set consisting of a 365 nm excitation and a 397 nm emission bandpass filter. NAD(P)H autofluorescence was recorded over the complete left liver lobe, allowing at least 15 different fields of view. Non-viable hepatocyte nuclei were labelled in vivo with an i.v. bolus of the vital dye PI (0.05 mg/100 g). As previously stated [21], PI-labelled nuclei were used to identify lethally injured hepatocytes. The fluorescent labelling of these nuclei was viewed using the 20x objective lens and a filter set with a 510 to 560 nm excitation and an emission barrier filter greater than 590 nm. Quantification of redox state and cell death was performed off-line by frame-by-frame analysis of the videotaped images using Meta Imaging Series Software (Ver. 6.1; Universal Imaging Corp., Downington, PA, USA). NAD(P)H fluorescence was densitometrically assessed and expressed as "average intensity/liver acinus". Gain, black level and enhancement settings were identical in all experiments. PI-labelled hepatocytes were expressed as number of cells/10-1 mm3.

Remission spectroscopy Hepatic sinusoidal HbsO2 was measured using the remission spectroscopy system Oxygen-to-See (O2C-ATS) supplied with the micro probe VM-3 (Lea Medizintechnik GmbH, Gieen, Germany). White light was continuously emitted via one channel of the micro probe light-guide and was continuously detected via another channel (channel diameter 70 m). The backscattered light was analyzed in steps of 1 nm (500650 nm). Each HbsO2 value was defined by specific Hb spectra. The local tissue light absorbance depends on the total local tissue content of Hb. The local content of Hb was calculated from the local light absorbance and emission. The flexible VM-3 micro probe allowed the detection of oxygen saturation of the left liver lobe placed on the glass slide of the inverted microscope. A special clamping system fixed the micro probe close to the surface of the glass slide and permitted contact-free systematic scanning of the liver lobe (Figure 6). At least 35 different observation points per animal were randomly chosen and examined. Before each experiment, the white standard of the micro probe was calibrated according to the technical instructions of the manufacturer. Measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels Blood was collected immediately after the microscopy procedure, via cardiac puncture. Blood samples were centrifuged at 6500 g, for 5 min, and the remaining serum analyzed, at 37C, by means of standard enzymatic techniques.

glass slide

left liver lobe

microscope stage

micro probe
Figure 6 Illustration of the experimental setup Illustration of the experimental setup. The flexible probe of the remission spectroscopy system was fixed on a special shaped clamp holder, which allowed the contact free scanning of the left liver lobe from the bottom side of the glass slide. The setup permitted systematic in vivo scanning of the liver sinusoidal HbsO2, without affecting the organ integrity.

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Blood gas analyses Blood samples for blood gas analyses were collected in heparinized syringes, via the insertion of a cannula in the left femoral artery, at the end of the microscopy procedure. The samples were immediately analyzed using the automated blood gas analyzing system Radiometer ABL 700 (Radiometer Medical Aps., Bronshoj, Denmark). Statistical analysis Data in text and Tables is given as: Mean (SD). Statistical differences between groups and from baseline within each group were determined by ANOVA, followed by the Tukey post-hoc test. The Kolmogorov-Smirnov test was previously used to confirm the normal distribution of data. For checking the nature and extend of the relationship between two variables linear regression analysis was performed. All figures were generated with Sigma Plot (Ver. 8.0) and statistical analyses were performed using Sigma Stat software (Ver. 2.0; SPSS Inc.; Mnchen, Germany). Differences were considered significant for p < 0.05.

9. 10. 11.

12. 13.

14. 15.

16. 17. 18.

Authors' contributions

CW conceived the design of the study and conducted the laboratory experiments; RB drafted the manuscript and coordinated the study; AK assisted in technical questions. NR participated in design and coordination and OE participated in animal procedures and in drafting the paper. All authors approved and read the final manuscript.

19. 20.

21. 22.

Acknowledgments

This work was supported by the Interdisziplinre Zenrum fr klinische Forschung (IZKF) of the Julius-Maximilians-Universitt Wrzburg (C. Wunder).
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