2.1.

Lesion of the Motor Cortex

LSI and MRI data were collected in parallel from an adult macaque monkey (a male macaca fascicularis: Mk-JH), 7 years old and weighting 7 kg at the time of the cortical lesion. Additional MRI data were obtained from a second male macaque monkey (Mk-BI; 5 years old, 5 kg body weight). All procedures were conducted in accordance to the Guide for Care and Use of Laboratory Animals (ISBN 0–309-05377–3; 1996) and approved by local veterinary authorities. As previously reported,¹¹ the monkeys were housed in our animal facilities in rooms of 12 m³, in which usually two to four monkeys were free to move and to interact among each other. (A new Swiss regulation was introduced in September 2010 requesting a volume of 45 m³ at least to be given to a group of up to five macaque monkeys.) The monkeys had free access to water and were not food deprived.

To perform the lesion of M1 in both monkeys (Mk-JH and Mk-BI) and, subsequently, to measure cerebral blood flow with LSI in Mk-JH, the animals were subjected to the following surgical procedures. The monkeys were first tranquilized with ketamine (Ketalar®; Parke-Davis, 5 mg/kg, intramuscularly); atropine was injected (0.05 mg/kg, intramuscularly) in order to reduce bronchial secretions. Before surgery, the animals were treated with the analgesic Carprofen (Rimadyl®, 4 mg/kg, subcutaneously) and the antibiotic Albipen® (Ampiciline 10%, 30 mg/kg, subcutaneously). Subsequently, Mk-JH and Mk-BI were anaesthetized with intravenous perfusion of 1% propofol (Fresenius®) mixed with a 5% glucose solution (1 volume of propofol and 2 volumes of glucose solution); ketamine was added to the perfusion solution (65 mg/100 ml). To prevent edema, Methylprednisolone (Solu-medrol, Pfizer®) was added to the propofol/glucose solution (1 mg/ml). The level of anesthesia was kept at an optimal level with a perfusion rate of the propofol/glucose mixture of 0.1 ml/min/kg. All surgeries were performed under sterile conditions. Heart rate, respiration rate, expired CO₂, arterial O₂ saturation, and rectal temperature were monitored throughout the surgery. After surgery, the monkey received Carprofen (pills of Rimadyl® mixed with food) daily and Albipen® (subcutaneously; same dose as above) every two days during one to two weeks.

A squared osseous sector of about 30 × 30 mm was opened above the central sulcus, centered at a medio-lateral coordinate (15 mm from midline) corresponding to the expected position of the hand area in M1. The dura-mater was incised and reclined in order to expose the central sulcus as well as the precentral and postcentral gyri. In monkey Mk-JH, ibotenic acid (Sigma 95%) was injected at six sites in the precentral gyrus, at a position roughly corresponding to the hand area based on its coordinate and the shape of the central sulcus at this location. The extent of the cortical territory, in which ibotenic acid was injected in Mk-JH, corresponded to the area of cortex captured by the charge-coupled device (CCD) camera for LSI measurements (Fig. 1). Acquisitions of cerebral blood flow with LSI (see below) were performed before and then after each individual injection of ibotenic acid. A volume of 3 μl of ibotenic acid solution (10 μg/μl in phosphate-buffered saline) was injected at each site by using a Hamilton microsyringe, positioned at 2 mm below the pial surface (Fig. 1). The total volume of ibotenic acid injected in Mk-JH was 18 μl, an amount representative of volumes injected in monkeys used for behavioral studies.^{4, 11, 12} In Mk-BI, from which only MRI data were derived, ibotenic acid was injected at 29 sites defined based on intracortical microstimulation,¹¹ at which a volume of 1 μl was infused (total amount 29 μl). The volume of ibotenic acid injected was larger in Mk-BI, because this animal was involved in the behavioral protocol,¹² requesting a lesion affecting the entire hand representation in M1. In contrast, Mk-JH was exclusively involved in the present LSI protocol and, therefore, the lesion was adapted to the cortical territory covered by the CCD camera rather than covering the entire hand representation in M1. In addition, in Mk-JH the infusion sites were somewhat more distant than in Mk-BI, and therefore, the volume injected at each infusion site in Mk-JH was larger. At the end of this experimental session aimed at lesioning the motor cortex, the dura mater was put back in place and sutured. The craniotomy was not closed. The muscle and skin were then sutured.

Fig. 1

(a) Setup of the LSI experiment, schematized over a standard lateral view of the left hemisphere of the macaque monkey. CS = central sulcus; AS = arcuate sulcus; PS = principal sulcus. (b) Time integrated image of the cortical surface, taken with the CCD camera. A vertically oriented blood vessel on the right follows the central sulcus, whereas another blood vessel of large diameter is horizontally oriented over the pre-central gyrus. The image was taken when the needle of an Hamilton syringue was inserted into the motor cortex, rostral to the central sulcus, to perform one of the six penetrations along which ibotenic acid was infused.

2.2.

Laser Speckle Imaging

2.2.1.

Data acquisition and image processing

For each subsequent LSI recording session conducted in Mk-JH only (at 1 and 3.5 weeks post-lesion, also under anesthesia as described above for the initial session), the skin was incised, the muscles reclined, and the dura was reopened as described above. For such a time interval between sessions, there were no adhesions between the dura and the blood vessels. A selected part of the surface of the cerebral cortex was homogeneously illuminated with a 785 nm single frequency laser (Toptica®, Munich, Germany), feeding a single mode fiber attached to a collimating lens (Schaefter + Kirchhoff®, Hamburg, Germany; Fig. 1). The whole illuminating device and the CCD camera have been positioned using a stereotaxic frame. The total laser power incident on the brain cortex was about 3 mW distributed over an area of several cm², thus preventing any physiological effects or superficial heating of cortical tissue. The diffuse reflected light was monitored in the image plane in the crossed polarization channel with a CCD camera (PCO Pixelfly, 640 × 480 pixels, 12 bit, exposure time 12 ms). A region of 10 mm by 7.5 mm of the cortex was imaged onto the 1/2^″ CCD chip at 0.64× magnification.

Individual measurements of 15 s duration at 50 frames per second (total 750 frames) have been streamed at full resolution to the hard disk using the Streampix 3 software package (NorPix Inc.®, Montreal, Quebec, Canada). The local speckle contrast K was computed from a b × b square of pixels and typically 40 successive frames,^{24, 32, 38} setting the actual time resolution of the experiment to 0.8 s. The image quality was further improved by implementation of a sliding window average (Figs. 2, 3), where essentially all camera pixels are replaced by a metapixel (value K). Only a few pixels at the image border need to be excluded from the final image due to the lack of sufficient neighbors. For a raw image with 640 × 480 pixels, and typically b = 5, this procedure provides a speckle image with a nominal resolution of 636 × 476 pixels (Fig. 3). The Labview® and Matlab® source codes are available free of charge from the authors (with no support) at http://physics.unifr.ch/en/page/54/.

Fig. 2

(a) Standard (temporal) LSI scheme. (b) High resolution LSI introducing a sliding window in space.

Fig. 3

Typical LSI in our experiment is constructed from 40 individual frames (resolution 640 × 480 pixels) and a spatial average of 5 × 5 camera pixels is performed at each point resolution in a final image with a nominal resolution of 636 × 476 pixels. Laser speckle images of the cerebral motor cortex calculated with different parameter settings: spatial averaging box size is set to 5² pixels for (a), (b), and (c); for (d) it is 2² pixels. (a) No time average, single frame, standard resolution scheme. (b) Time averaging over 40 frames, standard resolution scheme. (c) Time averaging over 40 frames, high resolution scheme (sliding window). (d) Time averaging over 250 frames, high resolution scheme (sliding window). The number of raw pixels used for the calculation of one LSI metapixels is 1000 for (b), (c), and (d) (example: box size * frames = 5² pixels * 40 frames = 1000 pixels). Axes labeling: x-y display the camera pixel at 0.64 magnification. The speckle contrast is color coded as shown by the color bar on the right of each LSI.

2.2.2.

Heartbeat filter

During the experiment the recorded image sequences exhibited a small periodic movement of the cerebral cortex due to the heartbeat/blood pressure variation of the animal. This movement leads to a measurable change in contrast (roughly +/− 10%) and in turn slightly blurs the image, when averaging over a sequence of images. To minimize this effect, we have implemented a software filter that allowed us to select images at a certain reference point in the heart beat cycle (Fig. 4). As expected, we found that the contrast time course essentially follows the heart beat with a frequency of approximately 80 beats per minute. A high pass filter was applied to select only the frames of the sequence with an average region of interest (ROI) contrast higher than a specified threshold. All other frames were discarded for the processing of the laser speckle image, thus roughly reducing the number of analyzed raw images by a factor of four to five. As can be seen in Fig. 4, this procedure allows us to reduce the systematic error due to heartbeat from more than 10 to about 3%.

Fig. 4

(a) Heart beat signal computed from the average contrast in a ROI of 100 × 100 pixels from successive individual frames. The data were normalized and baseline corrected. The green line shows the minimum threshold, all other frames below that line will not be used to calculate a high resolution contrast image. (b) LSIs of the cerebral motor cortex calculated from 5² pixels and 40 successive frames. The ROI analyzed for the heartbeat correction is indicated by the black square.

Residual movements of the skull were observed leading to minute shifts in x-/y-directions during a long time recording sequence. It was not straightforward to compensate these movements within our measurement scheme. The presence of these small drifts thus imposes a limit to the total recording time, and therefore, the statistical accuracy of the measurements, in order to avoid blurring effects. Here, we have chosen the image acquisition time such that the influence of these drifts is negligible.

2.3.

Magnetic Resonance Imaging Acquisition

The MRI data were acquired in Mk-JH and Mk-BI using a 1.5 Tesla Siemens© Symphony magnetic resonance scanner. To allow direct comparison of the extent of the cortical lesion at regular time points after the lesion, the anesthetized animal was placed in a decubitus ventral position with his head stabilized into a nonferromagnetic fixation frame with ear, mouth, and eye bars. MRI was performed under heavy sedation induced with subcutaneous injections of ketamine (Graeub® 10 mg/kg) associated with medetomidine (Graeub® 1 mg/kg). At the end of the acquisition, the anesthesia was reversed with Atipamezol (Pfizer®, 0.25 mg/kg). The acquisition parameters of the MRI data were the following: total of 19 images, slice thickness 2 mm, 2 TSE (TR 4500 ms and TE 129 ms), field of view 107 mm × 140 mm.

2.4.

Necropsy and Histology

At the end of the experiment, the monkeys were sacrificed^{5, 7} for histological analysis of the lesion. Mk-JH was sacrificed a few days after the last LSI session, whereas Mk-BI was sacrificed several months post-lesion as it was involved in a study of functional recovery.¹² The monkeys were sacrificed under deep anesthesia [initiated first with an i.m. ketamine injection followed by an i.p. lethal dose of sodium pentobarbital (90 mg/kg)] by transcardiac perfusion with 0.9% saline (400 ml) continued with fixative (three liters of four paraformaldehyde in 0.1 M phosphate buffer, pH = 7.6) and solutions (two liters each) of the same fixative containing increasing concentrations of sucrose (10, 20, and 30%). The brain was removed, dissected, and stored in a sucrose solution (30%) for 1.5 to 2 weeks. Frozen sections of the brain were then cut in the frontal plane at a thickness of 50 μm. Eight series of sections were collected with a cryotome (HM560, MICROM®, Switzerland). Among these series, one was Nissl-stained and one was immunocytochemically treated (SMI-32 antibody against a non-phosphorylated neurofilament epitope), as previously reported.^{7, 39} Furthermore, additional series were processed for other markers, such as Neuronal Nuclei [(NeuN) neuronal marker] and glial fibrillary acidic protein [(GFAP) glial marker], following previously described protocols.⁴⁰

3.1.

Injections of Ibotenic Acid

In the first LSI recording session conducted in Mk-JH, LSI acquisition was immediately made after incision of the dura in order to establish the cerebral blood flow level corresponding to the reference state under stable propofol anesthesia. Then, this first session comprised the procedure of ibotenic acid infusion in the motor cortex in order to produce a permanent cortical lesion, comparable to lesions made in the course of previous studies in our laboratory.^{4, 11, 12} Six penetrations with the needle of a Hamilton syringe were performed (Fig. 5), in the part of cerebral cortex rostral to the central sulcus, corresponding to M1.

Fig. 5

(a) Lateral view of the left hemisphere of Mk-JH, showing the cortical territory (circle) in which a lesion of the motor cortex was performed with infusion of ibotenic acid along six penetrations in the pre-central gyrus. The ROI is presumably located in the zone corresponding to the hand area. (b) CCD image (slightly tilted to the left) of the cortical surface in the pre-central gyrus, with location on the cortical surface of the six syringe penetrations (one to six). Same orientation as in the right panel of Fig. 1.

3.2.

Speckle Contrast Imaging (Laser Speckle Imaging)

The LSI in the reference state exhibited a high contrast [Figs. 6a and 7], corresponding to a moderate blood flow. As expected, the infusion of ibotenic acid at the first site of injection (Fig. 5) produced an immediate decrease of contrast, in line with an increase of cerebral blood flow (Fig. 7). Note, however, that the contrast slightly increased in the few minutes following the infusion of ibotenic acid at one site, as shown by the three LSI data points taken at 1.5 min intervals following the first post-infusion LSI acquisition. The same time course of contrast change was observed at all six injection sites during the few minutes following the actual infusion [Fig. 7a]. The next infusion at sites #2 and #3 produced a further decrease of contrast (increase of blood flow) [Fig. 7a]. As of the fourth injection site, the contrast reached a stable lowest level (maximal blood flow), although the small rebound of contrast was still present in the few minutes following each individual injection [Fig. 7a]. LSI was acquired during 25 more minutes after injection at the last site (site #6), taken at roughly 5 min intervals: there was a slow increase of contrast, finally reaching a stable level after about 75 min, situated in between those observed after the second and third injections of ibotenic acid. This contrast level at plateau is illustrated for the ROI in Fig. 6b, showing that it was clearly diminished as compared to the reference state [Fig. 6a].

Fig. 7

Time course of maximum, minimum, and average ROI contrast on (a) the day of ibotenic acid injection in Mk-JH as well as (b) at two further distant time points. In (a), the data points related to the six infusions of ibotenic acid were acquired after offset of the injection (the needle was removed from the tissue). In (b), the data point at reference and at one hour post-lesion corresponds to the data point at reference and at 100 min in the upper panel (errors bars were estimated from the time course of the measurements). The data at one and 3.5 weeks were derived from six and five independent measurements, respectively.

At that step, the dura was sutured (together with muscles and skin), the anesthesia was discontinued and the monkey was returned to the animal room. LSI was reacquired one week later and the contrast level was comparable to that observed about one hour after lesion [Figs. 6c and 7b]. The next LSI data point was acquired 3.5 weeks post-lesion, showing a dramatic increase of contrast, reaching a value higher than the initial reference value [Figs. 6d and 7b]. These data are indicative of a substantial decrease of blood flow at 3.5 weeks post-lesion, in line with the notion that ibotenic acid infusion produces a permanent lesion of the cortical tissue.

3.3.

Comparison of Laser Speckle Imaging, Magnetic Resonance Imaging and Histology

LSI and MRI were not acquired the very same day as the two facilities are located at distant sites. The first MRI image [Fig. 6e] was taken two days before the first LSI recording acquisition [Fig. 6a], both representing the reference state pre-lesion. The second MRI image was acquired one day after the first LSI session during which the ibotenic acid was infused at six sites. The zone of infusion corresponds to a marked hypersignal [red circle in Fig. 6f] present in the pre-central gyrus. The hypersignal in the MRI was found to be maintained in the same area [red circle in Fig. 6g] one week later, in parallel to the decrease of contrast in the LSI [Figs. 6c and 7b], associated to an increase of cerebral blood flow. The last MRI image was taken 3.5 weeks post-lesion [Fig. 6h], showing a complete disappearance of the hypersignal, in parallel to the dramatic decrease of cerebral blood flow at the same time point, reflected by the increase of LSI contrast [Figs. 6d and 7b]. The LSI and MRI signals in the ROI follow a parallel time course, at least for the four time points considered in the present study (reference, briefly after lesion, one week post-lesion, and 3.5 days post-lesion). The lesion generated by ibotenic acid infusion in the motor cortex is visible with both LSI and MRI only during a few days (at least one week). Later on, after 3.5 weeks, the signals in both LSI and MRI returned in the direction of the reference appearance pre-lesion [compare Figs. 6a and 6d for LSI; Figs. 6e and 6h for MRI], although the LSI signal had a higher contrast, corresponding to a decrease of cerebral blood flow. Evidence for the presence of a permanent cortical lesion was provided by the histology post-mortem for Mk-JH (Fig. 8). As shown in SMI-32 stained material (Fig. 8), the infusion of ibotenic acid produced a clear disruption of the cortical layers in the lesion site (delineated with the dashed line) without SMI-32 labeled pyramidal cells, whereas in the intact tissue adjacent to the lesion territory the cortical layers III and V are clearly visible by the presence of pyramidal neurons stained with SMI-32 (panels A1 and A2 in Fig. 8 at higher magnification). The appearance of the lesion is similar to that previously shown in monkeys subjected to such lesion and involved in our functional recovery protocols.¹¹ The focal cortical lesion does not generate a cavity in the gray matter but, as seen on Nissl staining, it corresponds to a homogeneous territory with loss of neuronal cell bodies in all cortical layers (Fig. 8, panels B1 and B2; comparable to a previous description in the rat: see Fig. 1 in Leroux ⁴¹). Furthermore, the lesioned territory seen with the above two markers (SMI-32 and Nissl) matches the zone corresponding to an interruption of the neuronal marker NeuN and, in contrast, an increase of the glial marker GFAP (Fig. 8, middle inset). The MRI data obtained in Mk-BI (not shown) exhibit a similar time course of the hypersignal associated to the ibotenic acid lesion in M1, as illustrated for Mk-JH in Fig. 6.

Fig. 6

LSI [(a),(b),(c),(d)] and MRI [(e),(f),(g),(h)] are compared in Mk-JH within a time frame of 3.5 weeks; (a), (e) reference before the injections; (b), (f) LSI taken 60 min post-lesion, MRI at one day post-lesion; (c), (g) LSI and MRI at one week post-lesion (one day apart from each other); (d), (h) LSI and MRI 3.5 weeks post-lesion (one day apart from each other). The highlighted area in the LSI figures shows the ROI wherein the average contrast was calculated for the quantitative data shown in Fig. 7. Calculation parameters for the LSI: 250 frames out of 750, HBC filter, sliding box, box = 5² pixels. The left and right MRI images on the same horizontal panel are taken at two rostro-caudal levels, distant by 2 mm, intercepting the ROI in the motor cortex.

Fig. 8

Histological assessment of the permanent lesion in the motor cortex in Mk-JH, on a frontal section of the motor cortex. Panel A is a low magnification of the lesion area, as seen with the marker SMI-32. In the top row, the panels A1 and A2 are higher magnification of the lateral and medial edges of the lesion territory, respectively, also in SMI-32 material. The damaged territory corresponds to an interruption of the layers III and V pyramidal neurons stained with SMI-32. In the middle, panel B is a low magnification in Nissl material of the lesion area. The panels B1 and B2 in the bottom row show at higher magnification the edges of the lesion with Nissl staining. In all panels, the dashed line delimits the lesion territory. The open arrow in panel B1 points to large Nissl stained neurons in layer V in an intact territory located slightly more lateral than the lesion. The inset in the middle at the right is a low magnification view of the lesion (white arrow), as seen in a combined NeuN (red) and GFAP (green) stained material. The corresponding lesion area is shown for GFAP labeling alone (in green), next to the white arrow. CS = central sulcus.

3.4.

Comparison of the Lesion Size Assessed with Magnetic Resonance Imaging, or Post-mortem on Histological Sections or with Laser Speckle Imaging

Based on consecutive histological sections as the one illustrated in Fig. 8 and using an ad-hoc function of the Neurolucida software (based on the Cavalieri method) as previously described,¹¹ the volume of the histological permanent lesion affecting the gray matter was estimated to be 18.8 mm³ in Mk-JH (after sacrifice, one month post-lesion). For comparison, the volume of the lesion as reflected by the hypersignal in the MRI scan (computed with the software OsiriX®) was 436 mm³ at one day post-lesion, 405 mm³ after one week, and 341 mm³ after two weeks. After 3.5 weeks, the lesion was no longer visible on the MRI scan [Fig. 6h]. In the second monkey (Mk-BI), the volume of the lesion assessed based on the hypersignal on the MRI scan was 262 mm³ one day post-lesion, 146 mm³ one week after lesion, 135 mm³ two weeks after lesion, and 97 mm³ three weeks after the lesion, whereas, 10 months post-lesion, the volume of the permanent lesion was estimated at 20.1 mm³ on SMI-32 stained histological material.

As expected, mostly due to edema in the days following the cortical lesion, the volume of the lesion derived from the MRI scans is clearly larger than the final volume histologically determined, by a factor of about 10 to 20. The difference may also comprise a deviation between the two methods to measure the volume of the lesion: although the infusion was aimed to the gray matter, some spread to the white matter is likely, which may be detected on the MRI scans, whereas the histological assessment was limited to the gray matter.

Finally, for comparison, the volume of the lesion was tentatively calculated from the LSI data at the time point at which a territory with reduced blood flow was observed, corresponding to the lesion. Based on an estimation of the surface of the cortical territory with reduced cerebral blood flow as seen after 3.5 weeks in Mk-JH [Fig. 6d] and considering an average cortical thickness of 2 mm, the volume of the cortical lesion as derived from LSI data is estimated to be around 58 mm³. Again, this figure is larger than the actual volume of the lesion histologically determined, but only by a factor of two to three in the comparison between LSI after 3.5 weeks and histology after four weeks. As the LSI approach does not permit an assessment of the cortical thickness in which cerebral blood flow is modified, one may compare in Mk-JH the extent of cortical surface exhibiting an increase of cerebral blood flow detected with LSI during the excitotoxic phase (immediately after infusion of ibotenic acid and one week later) with the cortical surface of the hypersignal observed from MRI one day and one week post-lesion. The cortical surface of the MRI hypersignal was 78.3 mm² at one day and 71.7 mm² at one week post-lesion, whereas the zone of increased blood flow corresponded to a cortical surface estimated at 22 mm², both on the day of ibotenic acid infusion and one week later.