Photobiomodulation inside the brain: a novel method of applying near-infrared light intracranially and its impact on dopaminergic cell survival in MPTP-treated mice

Laboratory investigation

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Object

Previous experimental studies have documented the neuroprotection of damaged or diseased cells after applying, from outside the brain, near-infrared light (NIr) to the brain by using external light-emitting diodes (LEDs) or laser devices. In the present study, the authors describe an effective and reliable surgical method of applying to the brain, from inside the brain, NIr to the brain. They developed a novel internal surgical device that delivers the NIr to brain regions very close to target damaged or diseased cells. They suggest that this device will be useful in applying NIr within the large human brain, particularly if the target cells have a very deep location.

Methods

An optical fiber linked to an LED or laser device was surgically implanted into the lateral ventricle of BALB/c mice or Sprague-Dawley rats. The authors explored the feasibility of the internal device, measured the NIr signal through living tissue, looked for evidence of toxicity at doses higher than those required for neuroprotection, and confirmed the neuroprotective effect of NIr on dopaminergic cells in the substantia nigra pars compacta (SNc) in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson disease in mice.

Results

The device was stable in freely moving animals, and the NIr filled the cranial cavity. Measurements showed that the NIr intensity declined as distance from the source increased across the brain (65% per mm) but was detectable up to 10 mm away. At neuroprotective (0.16 mW) and much higher (67 mW) intensities, the NIr caused no observable behavioral deficits, nor was there evidence of tissue necrosis at the fiber tip, where radiation was most intense. Finally, the intracranially delivered NIr protected SNc cells against MPTP insult; there were consistently more dopaminergic cells in MPTP-treated mice irradiated with NIr than in those that were not irradiated.

Conclusions

In summary, the authors showed that NIr can be applied intracranially, does not have toxic side effects, and is neuroprotective.

Abbreviations used in this paper:CPu = caudate-putamen complex; GFAP = glial fibrillary acidic protein; LED = light-emitting diode; MPTP = 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NIr = near-infrared light; PBS = phosphate-buffered saline; SNc = substantia nigra pars compacta; TH = tyrosine hydroxylase.

Object

Previous experimental studies have documented the neuroprotection of damaged or diseased cells after applying, from outside the brain, near-infrared light (NIr) to the brain by using external light-emitting diodes (LEDs) or laser devices. In the present study, the authors describe an effective and reliable surgical method of applying to the brain, from inside the brain, NIr to the brain. They developed a novel internal surgical device that delivers the NIr to brain regions very close to target damaged or diseased cells. They suggest that this device will be useful in applying NIr within the large human brain, particularly if the target cells have a very deep location.

Methods

An optical fiber linked to an LED or laser device was surgically implanted into the lateral ventricle of BALB/c mice or Sprague-Dawley rats. The authors explored the feasibility of the internal device, measured the NIr signal through living tissue, looked for evidence of toxicity at doses higher than those required for neuroprotection, and confirmed the neuroprotective effect of NIr on dopaminergic cells in the substantia nigra pars compacta (SNc) in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson disease in mice.

Results

The device was stable in freely moving animals, and the NIr filled the cranial cavity. Measurements showed that the NIr intensity declined as distance from the source increased across the brain (65% per mm) but was detectable up to 10 mm away. At neuroprotective (0.16 mW) and much higher (67 mW) intensities, the NIr caused no observable behavioral deficits, nor was there evidence of tissue necrosis at the fiber tip, where radiation was most intense. Finally, the intracranially delivered NIr protected SNc cells against MPTP insult; there were consistently more dopaminergic cells in MPTP-treated mice irradiated with NIr than in those that were not irradiated.

Conclusions

In summary, the authors showed that NIr can be applied intracranially, does not have toxic side effects, and is neuroprotective.

Many studies have shown that red to infrared light, in addition to its long-established ability to promote the healing of nonneural tissues, can be neuroprotective; that is, it can stabilize the CNS against stress from toxins, genetic mutation, or age.8,9,13 The first neuroprotective effects of near-infrared light (NIr), also termed “photobiomodulation” when the light source is a photodiode and “low-level laser therapy” when a laser source is used, occurred in the retina, showing protection of photoreceptors from toxic light or genetic stress.11,28 Subsequently, NIr treatment was shown to be neuroprotective in other systems. For example, NIr improves behavior and cellular function in experimentally induced stroke models in animals;10,21,29 in humans, NIr improves cognition and emotional function,3 as well as performance in a range of clinical tests after ischemic stroke,17 brain trauma,27 or depression.36

Near-infrared light treatment has also been shown to improve cell survival and locomotive behavior in Parkinson disease, in particular, in various animal models of the disease. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, NIr protects many dopaminergic cells in the substantia nigra pars compacta (SNc) from degeneration32,38 and restores functional activity of the subthalamic nucleus and zona incerta (key basal ganglia nuclei) to near control levels.37 Further, NIr treatment recovers various parameters of locomotive behavior, for example, mobility and velocity, in MPTP-treated mice.25,44 Moreover, in transgenic mice expressing the A53T human α-synuclein mutation, NIr treatment delays disease progression and reduces the severity of the disease phenotype.34 In addition to these in vivo studies are many in vitro studies that have shown that NIr protects rat striatal and cortical cells,22,46 as well as human neuroblastoma cells engineered to overexpress α-synuclein,34 against the toxic effects of MPTP. Furthermore, mitochondrial movement along axons in hybrid cells bearing mitochondrial DNA from patients with Parkinson disease improves substantially after NIr treatment, with movement restored to control levels.42

Notwithstanding these promising experimental results, as well as the serendipitous findings in a patient with Parkinson disease who underwent light treatment for a dental problem and then showed a reduction in parkinsonian signs,7 there have been no major clinical trials to date on the therapeutic effect of NIr in patients with Parkinson disease.20 One potential problem associated with this treatment relates to the effective and reliable penetration of NIr through the thick human cranium, meningeal layers, and brain parenchyma to reach the SNc in the midbrain. It is one thing for NIr to influence SNc survival 4–5 mm inside the skull of a mouse, and quite another to reliably influence SNc survival 80–100 mm inside the skull of a human. Indeed, almost all current clinical studies reporting the beneficial effects of NIr treatment have involved cases in which the target region was in the cortex, lying only 8–10 mm below the cranium,19 whether in patients suffering trauma,27 stroke,17,19 or depression.36 Structures lying much deeper in the human brain, such as in the SNc, may be largely beyond the range of externally applied NIr.

Several authors exploring the effectiveness of NIr treatment in different models of cerebral degeneration (for example, stroke) have investigated this issue. In rabbits, it has been estimated that NIr from a laser device may reach a depth of 25–30 mm from the cortical surface,21 while a similar estimate of approximately 20 mm has been predicted in humans.47 However, there would be considerable attenuation and loss of power intensity from the cranial surface and through the brain parenchyma. Shaw and colleagues38 have suggested that in mice, there is an approximately 90% decline in the NIr signal inside the cranium after external application via a light-emitting diode (LED). Similar sets of values have been recently reported for human cadaveric specimens.16 Further, Detaboada and colleagues10 estimated that there would be a power density decrease of more than 99% from the cortex to a depth of 18 mm in rats. Finally, in rats, Abdo and colleagues1 noted significant attenuation of the NIr signal, with power reducing approximately 90% at approximately 1 mm deep from the cortex.

All in all, these data indicate that NIr irradiation may not reach deeper brain regions in effective doses, particularly regions deeper than 20 mm from the cortical surface. This may present a limitation in the use of NIr as a long-term and reliable treatment in humans, particularly those suffering from Parkinson disease.19 Hence, there is a need to develop new and effective methods of delivering a strong NIr signal to deeper brainstem structures in humans. To this end, we here describe the use of a novel LED–optical fiber device made to deliver NIr intracranially, within the mouse brain. Specifically, we wanted to determine several issues related to this device: 1) feasibility, that is, whether the device was viable in the living brain ; 2) NIr signal attenuation within the living brain, that is, whether light levels changed at various distances from the source; 3) toxicity, that is, whether tissue necrosis occurred at the implant site; and 4) neuroprotection, that is, whether intracranially applied NIr, as with extracranially applied NIr,25,32,38 was neuroprotective to SNc dopaminergic cells after MPTP treatment.

Methods

Study Animals

Male BALB/c mice (n = 36) and Sprague-Dawley rats (n = 3) were used for these experiments. The mice were used for the feasibility, NIr signal attenuation, toxicity, and neuroprotection experiments using an LED–optical fiber. The rats, because of their larger brains, were used for the NIr signal measurements over a greater distance, as well as the toxic impact of a more intense NIr signal on brain tissue using a laser–optical fiber. Animals were housed on a 12-hour light/dark cycle with unlimited access to food and water. All experiments were approved by the Committee on the Ethics of Animal Experiments of the Grenoble University (ComEth).

Feasibility of the Device: Delivering NIr Into the Mouse Brain

LED–Optical Fiber

We developed an apparatus consisting of an optical fiber linked to an LED. A small, 3.5 × 2.7–mm 670-nm LED (SMT 670, Epitex) was aligned with and glued to an optical fiber (FT300EMT, Thorlabs) having a tip diameter of 300 μm (Fig. 1A–E). The step-index optical fiber was plastic coated to limit leakage of the NIr along the length of the fiber and to concentrate light at the tip (Fig. 1D). The LED was covered in black Glob Top epoxy glue (H20E, Epo-Tek) to absorb any excess light emitted by the LED (Fig. 1C). Electrical cables were attached to the LED and would subsequently be connected to a current regulator circuit and battery source (Fig. 2A). All LED–optical fibers were tested for power output using a calibrated light sensor (optical power meter, ML9002A, Anritsu). This ensured that each device emitted a similar output from the optical fiber, which was 0.16 mW (at 20 mA, maximum electronic power of the battery for this device). We preferred to use an LED instead of a laser device in these and most of the other experiments for several reasons: 1) it was more easily attached to the optical fiber, 2) the external WARP device used in previous studies25,32,38 was an LED (670 nm) and thus allowed us to make a direct comparison, and 3) we found that the LED emitted no heat across the implant and cranium and was satisfactory for use over longer periods (6 days).

Fig. 1.
Fig. 1.

Photographs of the LED–optical fiber device (A–E). A small 670-nm LED (arrowheads, A–D) was aligned with and glued to an optical fiber (arrows, A–E) with a tip diameter of 300 μm. The step-index optical fiber was plastic coated to limit leakage of NIr along the length of the fiber and to concentrate light at the tip (D, inset shows cross-section of the fiber). All LED–optical fibers were tested for NIr signal intensity using a calibrated light sensor; each device emitted a similar intensity of 0.16 mW of NIr signal. Diagram (F) showing the approximate region of device implantation within the lateral ventricle of the mouse brain (corresponding to Plate 37 from the atlas of Paxinos and Franklin31). Photograph (G) showing the experimental setup with a mouse in a stereotactic frame just prior to implantation of the optical fiber. Each LED–optical fiber was tested for efficacy for a few seconds just before implantation. Diagram (H) showing the outline of our first experiments testing the feasibility of the device. On Day 0, LED–optic fibers were implanted in the lateral ventricle of mice. Seven days later, the LED was turned on and ran continuously for 6 days. The appearance and behavior of the mice during this period was closely monitored.

Fig. 2.
Fig. 2.

Photographs of mice with NIr inside the brain. After the 7-day recovery period following surgery, the LED–optical fiber cables were attached to the batteries, and the LED was turned on for 6 days. The mice showed no visible discomfort or behavioral deficits with the intracranially applied NIr. The battery cables were long enough for the mice to move around the cage freely, with easy access to food and water.

Surgery

The skin and connective tissue overlying the cranium of the mice (n = 3) were pulled back. Stereotactic coordinates were used to place the optical fiber in the region of the lateral ventricle (Fig. 1F).31 We chose this site because it is easily targeted stereotactically (and it is relatively close to the SNc [4–5 mm] for use in the neuroprotection experiments described below). It presented the most convenient focus in the brain, underneath the skin, meningeal layers, and cranium. For device implantation, a small hole was made at a point 1.25 mm lateral and 0.7 mm posterior from the bregma, and the optical fiber was extended 2 mm ventral from the exposed cortical surface. A small screw was inserted in the opposite side of the cranium, and the region was filled with biological cement (a screw was used to secure the cement when dry). All devices were surgically implanted on the right side of the brain (Fig. 1G), and each LED–optical fiber was briefly tested just before implantation. Antiseptic was applied to the wound and the skin was sutured. The proximal ends of the battery cables were sutured so that they would remain securely in place within the subcutaneous tissue of the mouse (tunneling), whereas the distal ends were left free. Later, these distal ends were attached to a battery source placed on top of the mouse cage just after the first series of injections (see below). The battery cables were long enough for the mouse to move about the cage relatively freely (Fig. 2). Mice were allowed to recover for 7 days. The LED was not turned on during this recovery stage. On the 7th day postsurgery, the implanted LED was turned on and left on continuously for 6 days (Fig. 1H; the same period used for the neuroprotection experiments; see below). The overall appearance and behavior of the mice were closely monitored during this 6-day period.

Measuring the NIr Signal in the Living Rodent Brain

To determine the level of NIr attenuation in the living brain, we undertook two series of experiments. First, we measured the NIr signal emitting from the LED–optical fibers implanted in the lateral ventricle (same devices as those used for experiments described above) across different regions of the mouse brain (n = 3 mice), including the region of the SNc (Fig. 3). For these measurements, we used a light sensor (FDSP625 pigtailed photodiode, Thorlabs). We measured the NIr signal at 2 points in each of 3 trajectories through the brain; 2 tracts were in the vertical direction toward the region of the SNc (ipsilateral and contralateral sides; 1.25 mm lateral, 2.8 and 3.5 mm posterior from the bregma), and 1 tract followed an oblique trajectory at an angle of 32° to the LED–optical fiber (1.25 mm lateral, 2.8 mm posterior from the bregma). In a second series of experiments, we measured the NIr signal in the larger rat brain using a higher power output. We wanted to determine the strength of the NIr signal over a greater distance across the brain, approximately 10 mm in rats as compared with approximately 5 mm in mice. To this end, we used a laser device (Class IIIB, model IQ1A100, Power Technology) linked to an optical fiber; the fiber was implanted in the lateral ventricle, and measurements from 2 trajectories (ipsilateral and contralateral sides; 1.25 mm lateral, 5 mm posterior from the bregma) were made using the photodiode in a manner similar to that in the mouse. We used a laser device in these cases because it generated a higher power output than the LED, in fact, 2 orders of magnitude greater (67 mW compared with the 0.16 mW used in the main experiments). We found that, unlike the LED, the high power of the laser tended to emit heat across the implant and cranium, which was satisfactory for these measurement experiments and short-term exposures (2.5 hours; see below) but not for longer-term exposures (for example, 6 days). This heat would have caused the animals severe discomfort and stress.

Fig. 3.
Fig. 3.

Measurements of NIr signal from optical fibers implanted in the lateral ventricle in the rodent brain. Tissue section (A) showing the location of the different tracts of the photodiode (a, b, and c) from which measurements were taken. The NIr signal was measured at 2 points in each of 3 trajectories through the brain; 2 tracts were in the vertical direction toward the region of the SNc (one on the ipsilateral [a] and the other on the contralateral [b] side of the LED–optical fiber), and 1 tract followed an oblique trajectory at an angle of 32° to the optical fiber (c). The sagittal section corresponds to Plate 112 in the atlas of Paxinos and Franklin,31 and the adjacent diagram (left) of a dorsal view of a mouse's head shows the approximate location of the photodiode tracts in the cranium. Graph (B) of the percentage of NIr signal at different distances between the LED–optical fiber and the photodiode within the mouse (diamonds) and rat (circles) brains. There was a decline in the percentage of NIr signal with increasing distance between the optical fiber and the photodiode; the regression line (dotted line) was calculated using the formula 118.7 × exp(−1.097×) with R2 = 0.9883. We estimated that this decline in signal was 65% per mm of brain tissue. At 10 mm from the optical fiber, the NIr signal was less than 0.001% of that at the tip of the optical fiber in the lateral ventricle.

Toxicity: Impact of NIr Irradiation on Brain Tissue

LED–Optical Fiber Implant Sites

In each of the experimental groups used for the neuroprotection series of experiments (see below; Fig. 4), the histological appearance of the implant sites of the LED–optical fibers was examined. At the end of the neuroprotection experiments, mice from each group (5 per group, 30 total) were anesthetized with an intraperitoneal injection of chloral hydrate (4%, 1 ml/100 g). They were then perfused transcardially with phosphate-buffered saline (PBS) followed by 4% buffered paraformaldehyde. The brains were removed, postfixed overnight in the same solution, and placed in PBS with 30% sucrose added until the tissue block sank. The forebrain was then sectioned coronally and serially (at 50 μm; one in two series) using a freezing microtome. All sections were collected in PBS and immersed in a solution of 1% Triton (Sigma) and 10% normal goat serum (Sigma) at room temperature for approximately 1 hour. Sections (generally, 5 sections per animal) were processed for either tyrosine hydroxylase (TH) or glial fibrillary acidic protein (GFAP) immunohistochemistry. Sections were incubated in either anti-TH (1:1000, Sigma) or anti-GFAP (1:500, Sigma) for 24–48 hours at 4°C, followed by biotinylated anti–rabbit IgG (1:200, Bioscientific) for 2 hours at room temperature. For TH immunostaining, sections were immersed in streptavidin-peroxidase complex (1:200, Bioscientific) for 2 hours at room temperature and then reacted in a 3,3′-diaminobenzidine tetrahydrochloride (Sigma) PBS solution. Sections were mounted onto gelatinized slides, air dried overnight, dehydrated in ascending alcohols, cleared in Histo-clear, and covered with DPX. Most of the immunostained sections were lightly counterstained with neutral red as well. For GFAP immunostaining, sections were immersed in streptavidin–Texas Red complex (1:200, Bioscientific) for 2 hours at room temperature and then stained in DAPI. Sections were mounted in Fluoromount (Sigma) and viewed under a fluorescence microscope. For controls, sections were processed as described above, except that no primary antibody was used. These control sections were immunonegative.

Fig. 4.
Fig. 4.

Outline of the different experimental groups used for the neuroprotection series, namely Saline, Saline-pNIr, SalinecNIr, MPTP, MPTP-pNIr, and MPTP-cNIr. The LED–optic fibers were implanted in the lateral ventricles of mice on Day 0. Seven days later, the mice received 2 injections (saline or MPTP) over 24 hours. The mice had 4 NIr treatments (or none), which were administered immediately after each injection and about 6 hours later on the same day. After the fourth NIr treatment, mice were allowed to survive for 6 days thereafter. The LED was not turned on in the Saline and MPTP groups; hence, no NIr treatment was applied. The LED was turned on for 360 seconds total (4 × 90 seconds) in the Saline-pNIr and MPTP-pNIr groups and was left on continuously for the 6-day experimental period in the Saline-cNIr and MPTP-cNIr groups.

Laser–Optical Fiber Implant Sites

We also explored the impact of the higher power output laser–optical fiber (see above) on surrounding brain tissue in the rat brain. To this end, the laser–optical fibers were implanted within the caudate-putamen complex (CPu; 2.8 mm lateral, 1.7 mm posterior from the bregma, 4.5 mm ventral); the left side had no NIr signal (control), while the right side had a 67-mW power output. In the latter cases, the laser device was left on for 2.5 hours. These brains were fixed in aldehyde, sectioned horizontally, mounted on gelatinized slides (as described above), stained with cresyl violet, and then covered with DPX.

Neuroprotection of the SNc by NIr After MPTP Treatment

Experimental Design

For the neuroprotection series of experiments, we set up 6 experimental groups of mice (n = 30 mice, 5 mice per group; Fig. 4). They received intraperitoneal injections of either MPTP or saline, combined with simultaneous NIr treatments via LED–optical fibers or no light treatment. The different groups of mice were as follows: Saline (saline injections, no NIr), MPTP (MPTP injections, no NIr), Saline-pNIr (saline injections + pulsed NIr), MPTP-pNIr (MPTP injections + pulsed NIr), Saline-cNIr (saline injections + continuous NIr), and MPTP-cNIr (MPTP injections + continuous NIr). Our experimental paradigm of the simultaneous administration of parkinsonian insult and treatment was similar to that in previous animal models of Parkinson disease.23,25,33,37,38,43 This paradigm is unlike the clinical reality in which there is cell loss prior to therapeutic intervention; however, in our experimental study we hoped to determine the maximum effect of any NIr neuroprotection provided via the LED–optical fiber implant.

Surgery and MPTP Treatments

The LED–optical fibers were implanted in the lateral ventricles of mice as described above. Mice were allowed to recover for 7 days (Fig. 4), and the LED was not turned on during this recovery stage. On the 7th day postsurgery, mice received 2 MPTP (25-mg/kg injections for a total dosage of 50 mg/kg per mouse) or saline injections over a 24-hour period. This acute MPTP mouse model has been shown to induce a loss of dopaminergic cells in the SNc and has been used in previous neuroprotection studies.23,25,37,38 At 15 minutes and 6 hours after each injection, the LED was turned on in the mice in some of the experimental groups. In the Saline-pNIr and MPTP-pNIr groups, the LED was turned on for a 90-second pulse (same duration as in previous studies using the external WARP-LED apparatus),25,32,37,38 whereas in the Saline-cNIr and MPTP-cNIr groups, the LED was turned on and left on until the end of the experimental period (6 days). Following the experimental period, the mice were anesthetized, and their brains (midbrain sections including the SNc) were processed for TH immunocytochemistry as described above.

Cell Identification and Analysis

In this study, we used TH immunocytochemistry to describe patterns of cell death and protection. As in many previous studies, we interpreted a change in the TH+ cell number after experimental manipulation as an index of cell survival.5,23,25,32,33,38,43 If cells lose TH expression, they are likely to subsequently undergo death,5 which then leads to a reduction in the Nissl-stained (and TH+) cell number.23,43 Notwithstanding a small number of cells that may have a transient loss of TH expression,14 a key aspect of our study was whether NIr treatment preserved TH expression during a period when MPTP treatment alone would have abolished it.25,32,38 In terms of analysis, the number of TH+ cells within the SNc was estimated using the optical fractionator method (StereoInvestigator, MBF Science), as outlined previously.23,25,32,37,38,43 Briefly, systematic random sampling of sites—with an unbiased counting frame (100 × 100 μm)—within defined boundaries of the SNc was undertaken. Counts were made from every second section, and for consistency, the right side of the brain was counted in all cases. All cells (nucleated only) that came into focus within the frame were counted, and at least 5 sites were sampled per section. Individuals blinded to the different treatment groups undertook this analysis. For a comparison of cell numbers between groups, a 1-way ANOVA test (F and p values) was performed in conjunction with a Tukey-Kramer multiple comparison test (p value) using the GraphPad Prism program. Digital images were constructed using Adobe Photoshop (brightness and contrast levels were adjusted on individual images to achieve consistency, for example, in illumination, across the entire plate) and Microsoft PowerPoint programs.

Results

The results that follow will consider each of the following issues separately: 1) feasibility of the device in delivering NIr into the brain; 2) measuring the NIr signal in the living brain; 3) impact of NIr irradiation on brain tissue; and 4) neuroprotection of the SNc by NIr after MPTP treatment.

Feasibility of the Device: Delivering NIr Into the Brain

Our first experiments tested the feasibility of the new device, that is, whether intracranially applied NIr was viable in the living mouse brain (3 mice). Figure 2 features mice with LED–optical fibers implanted in the lateral ventricles and turned on. The NIr filled the cranial cavity and was evident through the skin and hair over the cranium and auricles and through the orbit and eyes. In these first cases, we left the device turned on for 6 days continuously (same time period as for the neuroprotection series; see below), and the appearance and behavior of the mice were monitored closely. In these mice, there did not appear to be any adverse effect from the intracranially applied NIr. They resumed normal activity, that is, eating and grooming, within a few hours after surgery and were never in any visible discomfort. Hence, according to these observations, the intracranially applied NIr appeared to be well tolerated by the mice, with no obvious signs of morbidity.

Measuring NIr Signal in the Living Brain

Figure 3 features the results of measuring the NIr signal from optical fibers implanted in the lateral ventricle in the brain (3 mice). Figure 3A shows the location of the different tracts of a photodiode from which measurements were taken, and the graph in Fig. 3B demonstrates the percentage of NIr signal at different distances between the LED–optical fiber and the photodiode within the mouse brain. In general, there was a gradual decline in the percentage of NIr signal as the distance between the optical fiber and the photodiode increased. We estimated that this decline in signal was 65% per mm of brain tissue. At 5 mm (Tract c within the contralateral midbrain) from the optical fiber, the NIr signal was < 1% of that at the tip of the optical fiber in the lateral ventricle. Of particular interest in this study was the level of NIr signal at the SNc. Here, we estimated that the NIr signal was, on the ipsilateral side (3.9 mm), approximately 2% (1.5–14.5 mW/cm2) and, on the contralateral side (4.7 mm), approximately 1% (0.5–7 mW/cm2) of that at the optical fiber in the lateral ventricle. Notwithstanding this large decline in signal across the brain, the NIr was clearly visible through the skin and hair of the mice (Fig. 2). Hence, even small traces of NIr signal were detectable with the human eye.

Figure 3B also features the percentage of NIr signal at different distances between the laser–optical fiber and the photodiode within the larger rat brain (3 rats). Overall, a similar pattern of decline occurred in the percentage of NIr signal as the distance between optical fiber and photodiode increased. At 10 mm from the optical fiber, twice the maximum distance in the mouse brain, the NIr signal was < 0.001% of that at the tip of the optical fiber in the lateral ventricle.

In summary, our measurements indicated that, despite the considerable diminution in the NIr signal across the brain (65% reduction/mm), the signal emitted from the optical fiber in the lateral ventricle did indeed reach the SNc region. In fact, we were able to detect an NIr signal up to 10 mm away from the optical fiber source.

Toxicity: Impact of NIr Irradiation on Brain Tissue

One of our major goals in this study was to determine whether the optical fiber itself and/or the intracranially applied NIr caused extensive damage to the brain parenchyma and surrounding tissue. We examined implant sites from TH-immunostained (Fig. 5A and B), DAPI-stained (to label cell bodies; Fig. 5C), cresyl violet–stained (to label cell bodies; Fig. 5E and F), and GFAP-immunostained (to label astrocytes; Fig. 5C′) sections.

Fig. 5.
Fig. 5.

Photomicrographs of implant sites in the lateral ventricle region of the mouse (A–C) and rat (E and F) brains. Typical examples of implant sites in the Saline (A) and Saline-cNIr (B and C) groups; similar patterns were seen in all the other groups (not shown). Tyrosine hydroxylase–immunostained sections (A and B), DAPI-stained section (C), GFAP-immunostained section (C′), and cresyl violet–stained sections (E and F). Sections featured in panels C and C′ are the same. The schematic coronal and horizontal sections correspond to Plates 37 and 19, respectively, in the mouse atlas of Paxinos and Franklin31(D) and indicate the approximate regions of implantation (optical fiber location in lateral ventrical [left] or CPu [red circles on right]) for the designated figures: panels A–C feature coronal sections (lateral to right, dorsal to top), and panels E and F feature horizontal sections (lateral to right, rostral to top). The mouse lateral ventricle is very thin; thus, our implant sites included regions of adjacent brain such as hippocampus (A and C) or CPu (B). The optical fiber tract was seen traversing the cortex and corpus callosum (arrowheads, A–C), and the site of the fiber tip usually had a characteristic square end within the brain parenchyma (arrows, A–C). Bar = 1 mm (A–C′) and 200 μm (E and F). cc = corpus callosum; Hc = hippocampal complex; LHS = left-hand side; RHS = right-hand side.

The main target for the implant site was the lateral ventricle, which lies between the cortex dorsally, hippocampus ventrally and medially, thalamus ventrally, and CPu laterally (Figs. 1F and 5D).31 The mouse lateral ventricle is very thin, and our implant sites included these adjacent regions. Figure 5 shows typical examples of LED–optical fiber implant sites in the Saline (Fig. 5A) and Saline-cNIr (Fig. 5B and C) groups; similar patterns were seen in the Saline-pNIr group and the corresponding MPTP groups (not shown). The fiber tract was seen traversing the cortex and corpus callosum, and the site of the fiber tip was found within the adjacent hippocampus (Fig. 5A and C) or CPu (Fig. 5B). The site of the optical fiber tip usually had a characteristic square end within the brain parenchyma (Fig. 5A–C).

From the TH-immunostained sections, we found no major differences in the implant sites of the different groups. In all the groups, there were no large zones of necrosis surrounding the LED–optical fiber tract and/or tip (Fig. 5A and B). In particular, in the cases in which the implant site was found within the CPu, there was no evidence of massive TH+ fiber degeneration in the regions surrounding the fiber tract and/or tip. In all cases, the only neural damage—or perhaps more appropriately, tissue displacement—appeared mechanical, caused by the fiber and/or the tip itself. Some regions of hemorrhage were seen occasionally within the implant site, but this was not limited to any group in particular (Fig. 5E and F). Hence, in the TH-immunostained sections, it appeared that NIr irradiation from the LED–optical fiber did not generate major necrosis of tissue. The only evident damage occurred because of the mechanical displacement of tissue caused by the fiber itself.

In the DAPI-stained sections, there was increased fluorescence on the borders of the LED–optical fiber tract and tip, indicating an increase in cell number (Fig. 5C). A similar pattern was evident in the GFAP-immunostained sections, with rich labeling around the implant site borders, presumably reflecting an increase in astrocytic glial cell activity and probably responsible for the increased DAPI labeling (Fig. 5C′). Such patterns were evident in all groups, and as with the TH immunostaining, we could discern no major differences in implant sites between the groups using DAPI staining and GFAP immunostaining. Hence, we concluded that the increase in glial activity on the implant site border was attributable to the mechanical damage caused by the optical fiber rather than the NIr irradiation.

To further investigate the impact of NIr light signal on brain tissue, we examined the histology of implant sites in cases that had a much greater NIr signal intensity, well above what was needed for neuroprotection (see below). In these cases, we used a laser–optical fiber that was able to generate 2 orders of magnitude greater power output than the LED–optical fiber (67 mW compared with the 0.16 mW used in the main experiments). Figure 5E and F shows cresyl violet–stained sections of the CPu on the control side (no NIr signal; Fig. 5E) and on the side receiving the high power output (Fig. 5F). In general, there was no difference between the histological appearances of the implant sites of the two sides, or between these and any of the other implant sites (see above). In these sections, as in those from the other implant sites, there was some hemorrhage within and gliosis surrounding the implant site.

In summary, there was little evidence of tissue necrosis caused by NIr irradiation, either in the pulsed (pNIr) or continuous (cNIr) groups of lower power output (LED 0.16 mW) or in the cases of higher power output (laser 67 mW). The only tissue damage appeared mechanical, caused by the optical fiber itself.

Neuroprotection of the SNc by NIr After MPTP Treatment

Figure 6 shows the estimated number of TH+ cells in the SNc in the 6 experimental groups. The LED–optical fiber devices were implanted in the lateral ventricle in the mice in each of these groups (Fig. 4). Overall, the variations in the number of cells between groups were significant (F = 4.6, p < 0.001, ANOVA). For the Saline, SalinepNIr and Saline-cNIr groups, the number of TH+ cells was similar; no significant differences were evident between these groups (p > 0.05, Tukey test). For the MPTP group, the TH+ cell number was reduced, compared with the Saline control groups (approximately 40%). These reductions were significant (p < 0.05). In the MPTP-pNIr and MPTP-cNIr groups, the TH+ cell number was higher than in the MPTP group, but slightly more so in the MPTP-pNIr (32%) than in the MPTP-cNIr (27%) group. This increase reached statistical significance for the MPTP-pNIr (p < 0.05, Tukey test) but not the MPTP-cNIr group. The number of cells in these two groups were not, however, significantly different from each other (p > 0.05, Tukey test). Unlike in the MPTP group, TH+ cell numbers in both the MPTP-pNIr and MPTP-cNIr groups were not significantly different from those in the saline groups (p > 0.05, Tukey test).

Fig. 6.
Fig. 6.

Graph showing the TH+ cell number in the SNc in 6 experimental mice groups. Bars show the mean ± standard error of the total number (on one side) in each group. Significant difference in number of TH+ cells, compared with the Saline group (†p < 0.001) and compared with the MPTP group (*p < 0.05). There were always more cells in the MPTP-pNIr and MPTP-cNIr groups than in the MPTP group, with the difference between the MPTP and MPTP-pNIr groups reaching significance. There was no significant difference in the number of cells in the MPTP-pNIr and MPTP-cNIr groups (p > 0.05).

These patterns are illustrated further in the Saline (Fig. 7A), Saline-pNIr (Fig. 7B), Saline-cNIr (Fig. 7C), MPTP (Fig. 7D), MPTP-pNIr (Fig. 7E), and MPTP-cNIr (Fig. 7F) groups. These figures show fewer TH+ cells in the SNc of the MPTP group than in the other groups. A key sign of an MPTP-induced lesion was a “bleached” or “washed-out” appearance in the SNc, a zone with few scattered TH+ cells. In the MPTP group, 80% of the SNc regions examined in the 5 mice had this appearance; the remaining 20% appeared similar to those in controls (that is, no bleached appearance). By contrast, in the MPTP-pNIr and MPTP-cNIr groups, only 20% and 30% of the SNc regions, respectively, had this appearance, with the rest of the groups similar to controls. These qualitative observations reflect the quantitative patterns described above.

Fig. 7.
Fig. 7.

Photomicrographs of TH+ cells in the SNc (lateral regions) of the Saline (A), Saline-pNIr (B), Saline-cNIr (C), MPTP (D), MPTP-pNIr (E), and MPTP-cNIr (F) groups. There were fewer TH+ cells in the SNc of the MPTP group than in the other groups. A key sign of an MPTP-induced lesion was a bleached appearance (region of few scattered TH+ cells; D) in the SNc. In the MPTP-pNIr and MPTP-cNIr groups, there were far fewer cases with this appearance; most cases had many TH+ cells similar to the number seen in controls. All panels feature coronal sections (lateral to right, dorsal to top). Bar = 100 μm. SNr = substantia nigra pars reticulata.

In summary, our results showed that there were more TH+ cells in the SNc of the MPTP-pNIr and MPTP-cNIr groups than in the MPTP group, indicating that NIr irradiation using the LED–optical fiber offered many SNc cells protection against MPTP toxicity.

Discussion

This is the first study documenting the impact of NIr on the brain when applied from inside the brain. We showed the feasibility of a new device that administered NIr intracranially. The device was well tolerated by the animals, with no observable adverse effects on their appearance and behavior. Our measurements of the NIr signal indicated that although the signal was substantially attenuated in brain tissue, it was traceable up to 10 mm from the optical fiber. There was no evidence of toxicity from the NIr, with no tissue necrosis evident at the implant site, even at high power levels. Finally, we showed that the intracranially applied NIr—as with extracranially applied NIr in previous studies—was neuroprotective to SNc dopaminergic cells after MPTP treatment. Each of these findings, together with the limitations of our study, will be considered in turn below.

Feasibility of a Novel Device: Intracranial Delivery of NIr

A major goal of this study was to test the feasibility of the new device, that is, whether intracranially applied NIr was viable in the living mouse brain. After 6 days of continuous NIr irradiation delivered via an LED–optical fiber, with the NIr filling the cranial cavity, there was no apparent adverse effect on the appearance and behavior of the mice; they were never observed to be in any discomfort. Furthermore, in a previous study in which NIr was applied intracranially—that is, as a means of localizing brain nuclei during functional neurosurgery in humans (via a stereotactic probe) and not for neuroprotective purposes—the authors concluded that NIr caused an extremely low level of morbidity.12 Only one clinical morbidity in over 200 probe insertions was reported in that study, and this single incident was presumably caused by damage to a perforating artery along the probe tract, not by the NIr itself.

Hence, from observations in mice (present study) and in humans in whom a localizing stereotactic probe was used,12 intracranially applied NIr does not appear to have any morbid side effects.

Measurements of the NIr Signal in the Living Brain

Our results showed that when NIr was applied from inside the rodent brain, it had an attenuation rate similar to that reported when it was applied from outside the brain.1,10,38 There was considerable diminution of the NIr signal across the brain; although we detected signal from 10 mm away from the source, the signal had reduced by over 99%. Overall, for both lower (0.16 mW) and higher (67 mW) power outputs, we estimated a 65% signal reduction across each millimeter of brain. Hence, over a distance of 80 to 100 mm—the predicted distance between any externally applied NIr from a WARP-LED to the SNc in humans—the NIr signal would be extremely weak, perhaps untraceable.21,47 These results make a strong case for the placement of an NIr–optical fiber source in regions near the SNc, perhaps within 5–10 mm, for diseased cells to receive sufficient signal and subsequent neuroprotection. We are currently undertaking our first implantations in the third ventricle in monkeys. This site lies close to the SNc (5–10 mm) and is used for deep brain stimulation.41

Toxicity of NIr: Evidence of Tissue Necrosis at the Implant Site?

Previous authors using external applications of NIr (for example, WARP-LED) at power intensities ranging from 7.5 to 75 mW/cm2 have reported no adverse effect on brain tissue;8,15,24,35,40 it is only at exceptionally high power intensities (750 mW/cm2) that some neuronal damage and negative behavioral outcomes have been noted.15 Notwithstanding this exceptionally high dose, the impact of NIr on all body tissues examined thus far has been overwhelmingly positive.8–10,13,19,24,34,35,40

In keeping with these previous reports, our histological results revealed little evidence of tissue necrosis caused by NIr irradiation when it was delivered intracranially and in direct contact with brain tissue, whether the radiation was low power (0.16 mW), pulsed (360-second exposure) or continuous for 6 days, or higher power (67 mW, 2.5-hour exposure). In each case, any tissue damage appeared mechanical, caused by the optical fiber itself. When relating our results to those of Ilic and colleagues,15 we may have needed to increase the power output at the optical fiber even more—perhaps by another factor of 10, which was well over the level needed for our positive neuroprotective result—to cause any neuronal damage and/or behavioral deficits.

Taken together, these data indicate that when NIr is applied at therapeutic doses, whether extra- or intracranially, it has little or no adverse effect on brain tissue. Future studies may extend these findings by exploring the longer-term effects, over months or years, of intracranially applied NIr.

Neuroprotection of Dopaminergic Cells in the SNc via NIr After MPTP Treatment

A major finding of this study was that NIr treatment, when applied from inside the brain, enhances dopaminergic cell survival in the SNc of MPTP-treated mice. There were more TH+ cells in the MPTP-NIr groups than in the MPTP group, indicating that NIr protected cells from the MPTP insult. This finding is consistent with those in several in vitro22,34,42,44,46 and in vivo25,32,34,38,45 studies that have documented better cell survival after the external application of NIr. In fact, the magnitude of neuroprotection was similar to those in previous studies using the same acute MPTP (50 mg/kg) model; whether applied externally25,38 or internally (present study), TH+ cell numbers in the MPTP-NIr groups were not only greater than in the MPTP group, but also very similar to those in the saline control groups. It should be noted, however, that we did find some evidence of degeneration in the MPTPNIr cases, indicating that NIr treatment may offer protection to many but not all of the dopaminergic cells. The number of degenerating cells must have been small, because no significant differences were evident between the MPTP-NIr groups and the saline controls.

We showed that TH+ cell survival was just as effective, if not marginally better (approximately 5%, although not significantly different), when NIr was applied in pulses (360 seconds, MPTP-pNIr group) rather than continuously (6 days, MPTP-cNIr group). Previous studies have also noted this feature; it has been reported that when NIr is applied by pulse, it is more effective in treating traumatic brain injury and stroke than if applied continuously.2,19 It appears that the beneficial effect of NIr has a threshold, and after a certain level of NIr stimulation, the effect tapers off.18 Its mechanism of action on mitochondrial activity, for example, has been likened to a “switch” (G. Jeffery, personal communication, 2013), but the mechanism of this switch is unknown. Our finding that NIr had beneficial effects on the survival of SNc cells over such a short pulse period is most encouraging for future endeavors in humans in that there would be less wear (and associated cost) on LED or laser devices and the batteries powering them. The shorter exposure periods would also make for apparently safer use in humans, although at the exposure times we used, we found no evidence of morbidity or tissue toxicity.

The mechanisms that protected cells from death after NIr treatment are unknown, but several can be suggested. First, NIr may have stimulated mitochondria in the SNc cells directly by increasing adenosine-5′-triphosphate (ATP) content, electron transfer in the respiratory chain, and activation of photoacceptors (for example, cytochrome oxidase), all leading to enhanced survival.4,30,45 Second, NIr may have also triggered a more indirect global systemic response.39 Several studies have reported remote, often bilateral, effects on body tissues after the local application of NIr to, for example, either skin wounds6 or the dorsum of the body.39 It has been suggested that NIr stimulates the immune system and that the cells of this system help protect nigral cells from MPTP insult.39 There is evidence that NIr treatment is associated with the downregulation of proinflammatory cytokines (γ−interferon, α−tumor necrosis factor) and the upregulation of antiinflammatory cytokines (IL-4, IL-10).26 Finally, several groups have suggested that NIr promotes functional recovery by stimulating neurogenesis and neuronal migration.8,19,29 It remains to be determined which of these mechanisms protects SNc cells, but our working hypothesis is that they are not mutually exclusive and that they all contribute to the process.39 We are currently exploring this issue of NIr mechanisms in other studies.

Limitations of This Study

Our study has several limitations. First, we tested the impact of NIr at two doses from a low power intensity (0.16 mW) via an LED; the doses were estimated at 1.5 mW/cm2 (pulse) and 14.5 mW/cm2 (continuous) at the ipsilateral SNc. We used this intensity because it was the maximum power available from the battery source. Fortunately, our two doses from this intensity provided a neuroprotective effect with no toxic impact on neural tissue. A higher power intensity was achievable—in fact 2 orders of magnitude higher—but we had to use a laser device. The device was satisfactory for short bursts of NIr exposure (for example, 2.5 hours) or for measurements of NIr across the brain, but because it generated heat across the implant site and cranium, it was not satisfactory for longer periods (for example, 6 days). Authors of future studies may explore a greater range of doses and intensities from better-developed LED and laser devices (for example, those that generate less heat). For these future studies, our results will serve as a primary template.

Second, we were unable to undertake a quantitative behavioral assessment of the mice in the different groups. We found that although the mice did not show any visible discomfort and moved across the cage freely, the surgery and optical fiber implants did impair a more detailed analysis of locomotor activity. We made some initial analyses of velocity, high mobility, and immobility in an open-field test25 but found that there were no significant differences in these locomotor activities across the different groups. This outcome was very different from the behavioral patterns reported in animals from the same groups (for example, Saline, Saline-NIr, MPTP, and MPTP-NIr) but with no surgical implants.25 Hence, we concluded that the surgical implants in the mice masked any differences in locomotor activity and subsequently decided that our behavioral observations in this study would be qualitative. A constructive quantitative analysis of locomotor activity would be possible on larger animals, such as monkeys, in which the surgical implants would be less intrusive and less likely to impair behavioral changes in the different experimental groups. We are currently exploring this issue.

Third, our results were based on shorter-term experiments, that is, over a period of 7 days. Although the results generated over this time provided key indications for device feasibility and toxicity to surrounding tissue, authors of future studies may consider longer-term experiments, perhaps in conjunction with a more chronic model of MPTP.32

Fourth, our study was undertaken in the rodent brain, an order of magnitude smaller than the human brain. Nevertheless, our primary aims were to test device feasibility, toxicity to tissue, and the measurement of NIr through living tissue, and for these purposes, the rodent brain was a valuable model. Further, for our neuroprotection study, we wanted to use a well-established parkinsonian animal model to test the cell-saving capabilities of internally applied NIr. The mouse provided us with such a model, one that previous studies have used to measure neuroprotection after externally applied NIr25,32,38 and serve as important points of comparison.

Notwithstanding these limitations, our results furnish key first insights into the feasibility and neuroprotective effects of intracranially applied NIr.

Conclusions

Our overall goal was to develop an effective and reliable method of applying NIr in animals with a larger brain and cranium (for example, monkeys and humans). To this end, we aimed to largely avoid problems of signal attenuation when delivery is extracranial and to develop a device that applies the NIr within the brain itself, in regions near the target diseased cells. Notably, we found that when NIr is applied from inside the brain and near the intended target region (SNc), it is well tolerated by the animal, does not generate local necrosis, and has sufficient power to protect neurons from toxic insult.

Acknowledgments

We are forever grateful to Tenix Corp., Salteri family, Sir Zelman Cowen Universities Fund, Fondation Philanthropique Edmond J Safra, France Parkinson, and the French National Research Agency (ANR Carnot Institute) for funding this work. We thank Sharon Spana, Rat Venceslas, Vincente Di Calogero, Christophe Gaude, Caroline Meunier, and Leti-DTBS staff for excellent technical assistance.

Disclosure

The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

Author contributions to the study and manuscript preparation include the following. Conception and design: Mitrofanis, Moro, Torres, Perraut, Benabid. Acquisition of data: Mitrofanis, Moro, El Massri, Torres, Ratel, De Jaeger, Chabrol, Perraut, Bourgerette, Berger, Purushothuman, Johnstone, Benabid. Analysis and interpretation of data: Mitrofanis, Moro, El Massri, Torres, De Jaeger, Chabrol, Perraut, Stone, Benabid. Drafting the article: Mitrofanis, Moro, Torres, Stone, Benabid. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Mitrofanis. Statistical analysis: Mitrofanis, Moro, Torres, Benabid. Administrative/technical/material support: Mitrofanis, Moro, El Massri, Torres, Ratel, Chabrol, Perraut, Bourgerette, Berger, Purushothuman, Johnstone, Benabid. Study supervision: Mitrofanis, Moro, Torres, Benabid.

This article contains some figures that are displayed in color online but in black-and-white in the print edition.

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Article Information

Address correspondence to: John Mitrofanis, Ph.D., Department of Anatomy and Histology F13, University of Sydney, Sydney 2006 Australia. email: john.mitrofanis@sydney.edu.au.

Please include this information when citing this paper: published online October 25, 2013; DOI: 10.3171/2013.9.JNS13423.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Photographs of the LED–optical fiber device (A–E). A small 670-nm LED (arrowheads, A–D) was aligned with and glued to an optical fiber (arrows, A–E) with a tip diameter of 300 μm. The step-index optical fiber was plastic coated to limit leakage of NIr along the length of the fiber and to concentrate light at the tip (D, inset shows cross-section of the fiber). All LED–optical fibers were tested for NIr signal intensity using a calibrated light sensor; each device emitted a similar intensity of 0.16 mW of NIr signal. Diagram (F) showing the approximate region of device implantation within the lateral ventricle of the mouse brain (corresponding to Plate 37 from the atlas of Paxinos and Franklin31). Photograph (G) showing the experimental setup with a mouse in a stereotactic frame just prior to implantation of the optical fiber. Each LED–optical fiber was tested for efficacy for a few seconds just before implantation. Diagram (H) showing the outline of our first experiments testing the feasibility of the device. On Day 0, LED–optic fibers were implanted in the lateral ventricle of mice. Seven days later, the LED was turned on and ran continuously for 6 days. The appearance and behavior of the mice during this period was closely monitored.

  • View in gallery

    Photographs of mice with NIr inside the brain. After the 7-day recovery period following surgery, the LED–optical fiber cables were attached to the batteries, and the LED was turned on for 6 days. The mice showed no visible discomfort or behavioral deficits with the intracranially applied NIr. The battery cables were long enough for the mice to move around the cage freely, with easy access to food and water.

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    Measurements of NIr signal from optical fibers implanted in the lateral ventricle in the rodent brain. Tissue section (A) showing the location of the different tracts of the photodiode (a, b, and c) from which measurements were taken. The NIr signal was measured at 2 points in each of 3 trajectories through the brain; 2 tracts were in the vertical direction toward the region of the SNc (one on the ipsilateral [a] and the other on the contralateral [b] side of the LED–optical fiber), and 1 tract followed an oblique trajectory at an angle of 32° to the optical fiber (c). The sagittal section corresponds to Plate 112 in the atlas of Paxinos and Franklin,31 and the adjacent diagram (left) of a dorsal view of a mouse's head shows the approximate location of the photodiode tracts in the cranium. Graph (B) of the percentage of NIr signal at different distances between the LED–optical fiber and the photodiode within the mouse (diamonds) and rat (circles) brains. There was a decline in the percentage of NIr signal with increasing distance between the optical fiber and the photodiode; the regression line (dotted line) was calculated using the formula 118.7 × exp(−1.097×) with R2 = 0.9883. We estimated that this decline in signal was 65% per mm of brain tissue. At 10 mm from the optical fiber, the NIr signal was less than 0.001% of that at the tip of the optical fiber in the lateral ventricle.

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    Outline of the different experimental groups used for the neuroprotection series, namely Saline, Saline-pNIr, SalinecNIr, MPTP, MPTP-pNIr, and MPTP-cNIr. The LED–optic fibers were implanted in the lateral ventricles of mice on Day 0. Seven days later, the mice received 2 injections (saline or MPTP) over 24 hours. The mice had 4 NIr treatments (or none), which were administered immediately after each injection and about 6 hours later on the same day. After the fourth NIr treatment, mice were allowed to survive for 6 days thereafter. The LED was not turned on in the Saline and MPTP groups; hence, no NIr treatment was applied. The LED was turned on for 360 seconds total (4 × 90 seconds) in the Saline-pNIr and MPTP-pNIr groups and was left on continuously for the 6-day experimental period in the Saline-cNIr and MPTP-cNIr groups.

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    Photomicrographs of implant sites in the lateral ventricle region of the mouse (A–C) and rat (E and F) brains. Typical examples of implant sites in the Saline (A) and Saline-cNIr (B and C) groups; similar patterns were seen in all the other groups (not shown). Tyrosine hydroxylase–immunostained sections (A and B), DAPI-stained section (C), GFAP-immunostained section (C′), and cresyl violet–stained sections (E and F). Sections featured in panels C and C′ are the same. The schematic coronal and horizontal sections correspond to Plates 37 and 19, respectively, in the mouse atlas of Paxinos and Franklin31(D) and indicate the approximate regions of implantation (optical fiber location in lateral ventrical [left] or CPu [red circles on right]) for the designated figures: panels A–C feature coronal sections (lateral to right, dorsal to top), and panels E and F feature horizontal sections (lateral to right, rostral to top). The mouse lateral ventricle is very thin; thus, our implant sites included regions of adjacent brain such as hippocampus (A and C) or CPu (B). The optical fiber tract was seen traversing the cortex and corpus callosum (arrowheads, A–C), and the site of the fiber tip usually had a characteristic square end within the brain parenchyma (arrows, A–C). Bar = 1 mm (A–C′) and 200 μm (E and F). cc = corpus callosum; Hc = hippocampal complex; LHS = left-hand side; RHS = right-hand side.

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    Graph showing the TH+ cell number in the SNc in 6 experimental mice groups. Bars show the mean ± standard error of the total number (on one side) in each group. Significant difference in number of TH+ cells, compared with the Saline group (†p < 0.001) and compared with the MPTP group (*p < 0.05). There were always more cells in the MPTP-pNIr and MPTP-cNIr groups than in the MPTP group, with the difference between the MPTP and MPTP-pNIr groups reaching significance. There was no significant difference in the number of cells in the MPTP-pNIr and MPTP-cNIr groups (p > 0.05).

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    Photomicrographs of TH+ cells in the SNc (lateral regions) of the Saline (A), Saline-pNIr (B), Saline-cNIr (C), MPTP (D), MPTP-pNIr (E), and MPTP-cNIr (F) groups. There were fewer TH+ cells in the SNc of the MPTP group than in the other groups. A key sign of an MPTP-induced lesion was a bleached appearance (region of few scattered TH+ cells; D) in the SNc. In the MPTP-pNIr and MPTP-cNIr groups, there were far fewer cases with this appearance; most cases had many TH+ cells similar to the number seen in controls. All panels feature coronal sections (lateral to right, dorsal to top). Bar = 100 μm. SNr = substantia nigra pars reticulata.

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