Protective Effects of Acetyl L-Carnitine on Inhalation Anesthetic-Induced Neuronal Damage in the Nonhuman Primate

Translational molecular imaging using positron emission tomography (PET) is a radionuclide imaging modality that enables direct in vivo measurements of multiple biological processes, disease progression and response to therapy in various organs. The development of microPET imaging applications has provided the ability to collect sensitive and quantitative three-dimensional molecular information from the living brains of a variety of animals including nonhuman primates (NHPs) [1-3]. Repeat studies with microPET imaging allow the adverse effects of toxic insults to be followed over time in individual animals that can also serve as their own controls [4-6]. For assessing the neurotoxic effects associated with developmental exposures to anesthesia, we have developed a microPET protocol to target neuronal damage in vivo using imaging approaches [7,8].

Exposure to general anesthetics during the period of rapid neuronal growth and synaptogenesis (the brain growth spurt period) in the developing animal brain can induce adverse effects including long-term neurotoxicity and undesired neurological consequence [9-13]. Nonhuman primates are exceptionally good animal models of neurodevelopment and for the detection of potential neurodegenerative effects associated with exposure to inhaled anesthetics. In our previous study [14], neonatal rhesus monkeys (PND 5 or 6) were exposed to a combination of 70% N₂O+1% ISO to achieve and maintain a surgical plane of anesthesia. Significantly elevated neuronal damage, as indicated by increased numbers of caspase-3-, silver stain-, and Fluoro- Jade C-positive cells, was observed in neocortical areas, especially in layers II and III of the frontal cortex, temporal gyrus and hippocampus. MicroPET scans using [¹⁸F]-radiolabelled]-N-(2-(2-fluoroethoxy) benzyl)-N-(4-phenoxypyridin-3-yl) acetamide) ([¹⁸F]-FEPPA) (6) demonstrated that anesthetic exposure induced significantly increased uptake of [¹⁸F]-FEPPA in the temporal lobe and frontal lobe of exposed animals in time dependent manner.

Acetyl-L-carnitine (ALC) (Figure 1a), an esterified compound of L-carnitine (4-N-trimethylamino-3-hydroxybutyric acid, LC) (Figure 1b), is essential for the β-oxidation of long chain fatty acids in mitochondria to generate ATP. Compared to LC, ALC is more actively transported across the blood-brain barrier and, thus, can be more effective in modulating brain metabolism [15-18]. It has been reported that ALC provides neuroprotective benefits in neurodegenerative and aging situations [19-26], although the mechanism of its neuroprotective effect is not known. The purpose of the current imaging study was to determine if co-administration of ALC could protect or reverse anesthetic-induced neurotoxicity.

Figure 1: Chemical structure of L-carnitine (LC) and Acetyl-L-carnitine (ALC).

Animals

All animal procedures were approved by the National Center for Toxicological Research (NCTR) Institutional Animal Care and Use Committee and conducted in full accordance with the PHS Policy on Humane Care and Use of Laboratory Animals.

All monkeys were born and housed at the FDA’s National Center for Toxicological Research nonhuman primate research facility. Animal procedures were designed to minimize the number of animals required and any pain or distress associated with the experimental procedures.

A total of sixteen (PND 5 or 6, 460-500 g) rhesus monkeys (Macacamulatta) were utilized. Seven male and nine female monkeys were randomly assigned to control (n=4, 2 males, 2 females), control+ALC (n=4, 2 males, 2 females), ISO+N₂O (n=4, 1 male, 3 females) and ISO+N₂O+aLc (n=4, 2 males, 2 females) groups. Immediately prior to the initiation of anesthesia or sequestration (controls), the neonates were separated from their anesthetized mothers, removed from their home cage and hand carried to a procedure room. Control animals were maintained in a holding cage other than the exposure chamber with water, but no food, and were not sedated for physiological measurements or blood sample collections. Animals in the anesthetic exposure groups were exposed to a mixture of 70% N₂O/29% oxygen and 1% ISO for 8 hours.

Experimental procedures

Nitrous oxide (N₂O) and oxygen were delivered using a calibrated anesthetic machine with blender (Bird Corporation, Palm Springs, CA, USA). Isoflurane (ISO) was delivered using an agent-specific vaporizer (E-Z Anesthesia, Palmer, PA, USA) attached to the anesthetic machine. To administer a specific concentration of N₂O/oxygen/ISO in a tightly controlled environment, an anesthesia chamber (Euthanex Corp. Palmer, PA) was used. Monkeys were kept in this chamber on a circulating water heating pad to maintain body temperature at approximately 37°C throughout the experiment. Stable gas and volatile anesthetic concentrations were attained in the chamber within 5 min of the start of exposure. For controls, room air was used in place of the inhalation anesthetics. For the exposure groups, the N₂O/oxygen/ ISO mixture was continuously delivered to the chamber for 8 hours. A relief valve on the anesthesia chamber allowed escape of gases to avoid accumulation of carbon dioxide and waste anesthetic gas was scavenged using an attached canister containing activated charcoal. For groups with ALC, acelyl-l-carnitine (Sigma, St. Louis, MO, USA) was i.p. administered at doses of 100 mg/kg in a saline vehicle, 1 hour before and 4 hours following the start of exposure to room air or the anesthetics.

During the experimental period, dextrose (5%) was administered by stomach tube (5 ml) every 2 h to both treated and control monkeys to maintain blood glucose levels. Glycopyrrolate (0.01 mg/kg, American Reagent, Shirley, NY, USA) was administered intramuscularly prior to exposures to both treated and control monkeys to reduce airway secretions. Once exposures were complete, animals were monitored in an incubator until complete recovery and then returned to their mothers (approximately 2 h after the 8 h exposures): control animals were kept in the exposure chambers under room air for a total of 10 h.

Monitoring of physiological parameters: The physiological parameters of the neonatal monkeys were monitored following procedures described previously [27,28]. Briefly, non-invasive pulse oximetry (N-395 Pulse Oximeter, Nellcor, Pleasanton, CA; MouseO_X Plus Vital Sign Monitor, Starr™ Life Sciences, Oakmont, PA), capnography (Tidal Wave Hand-held capnography, Respironics, Murrysville, PA), sphygmomanometry (Critikon Dynamap Vital Signs Monitor, GE Healthcare, Waukesha, WI, USA), and rectal temperature monitoringwere used to verify the physiological status of subjects. Heart and respiration rates, arterial blood O₂ saturation levels, expired CO₂ concentrations, and rectal temperatures, systolic and diastolic blood pressures were recorded every one or two hours in anesthetized and control animals, respectively. Venous blood (approximately 250 μl) was collected at two-hour intervals for measurement of plasma glucose concentrations, venous blood gases, pH, and hematocrits (GEM^®Premier™ 4000, Instrumentation Laboratory, Lexington, MA). Statistical analyses of physiological parameters were performed using Student’s t-test for comparisons between treated and control groups. One-way analysis of variance was used to evaluate the effect of time under anesthesia on body temperature. The null hypothesis was rejected at a probability level of P<0.05.

Radiotracer preparation: [¹⁸F]-FEPPA was prepared by 3D-Imaging LLC (Little Rock, AR) according to Wilson et al. [29] from the corresponding to sylate as described therein with minor modifications to the procedure. [¹⁸F]-FEPPA was produced in >98% purity at a specific activity EOS of 1.1-3.7 TBq (30-100 Ci)/μmole, as compared to the previously reported value of 11-37 MBq (0.3-1 Ci)/ μmole [30]. Specific activity at the time of use was one tenth to twothirds of the EOS value.

MicroPET: A commercial high resolution small animal PET scanner (Focus 220, Siemens Preclinical Solution, Knoxville, USA) was used to quantitatively acquire images of the monkey brain. The scanner has 96 lutetium oxyortho-silicate detectors and provides a transaxial resolution of 1.35 mm full-width at half-maximum. Data were collected in a 128×128×95 matrix with a pixel width of 0.475 mm and a slice thickness of 0.815 mm.

Computed tomography: Monkeys were also imaged in a newly developed mobile neurological CereTom CT scanner (Neurologica Corps. Danvers, MA, USA). The CT gantry of the CereTom scanner moves while the subject remains externally supported and fixed in space and therefore allows it to be physically connected to the microPET scanner. Exposure settings for each CT scan were 120 kVp, 5 mAs, scan time=120s. Data were collected in a 512×512 matrix with a pixel width of 0.49 mm and a slice thickness of 1.25 mm.

MicroPET/CT image acquisition: The first microPET and CT scans of each monkey brain weretaken on PND 6 or 7, the day following the experimental exposures on PND 5 or 6. Follow-up microPET/ CT scans occurred approximately one week (PND 14), three weeks (PND 30) and 5 months following treatment (at 6 months of age). For collection of the microPET/CT images, animals were positioned on a modified external bed controlled by the microPET unit. The bed replaced the standard microPET bed and allowed for sufficienttravel to move animals through the microPET and CT fields of view (over an axial range of greater than 50 cm). Throughout microPET/CT imaging sessions, monkeys were anesthetized with 1.5% isoflurane gas alone delivered through a custom face mask and an electronic heating pad was used to maintain body temperature at approximately 37°C. For each imaging session [¹⁸F]-FEPPA (56 MBq) was injected into the lateral saphenous vein of each animal (anesthetized). Immediately following the injection, a set of serial microPET images was collected to assess the influx of the tracer for 2 hours (24 frames, 5 min each). Micro-computed tomography (microCT) coronal images were obtained immediately after microPET imaging for the purpose of fusing anatomical (CT) with molecular (PET) data. The microCT images were acquired for 2 min and concurrent image reconstruction was achieved using 3D reconstruction software (ASIPro™; Concorde Microsystems, Inc, Knoxville, TN) installed in the CereTom CT controller unit. Once scanning was complete, animals were monitored in an incubator within a shielded isolation area until complete recovery (approximately 2 hours) and then returned to their mothers. Control animals were kept in the exposure chambers under room air for the same amount of time.

Imaging data analysis: Medical image analysis software, ASIPro? (Concorde Microsystems, Inc, Knoxville, TN) was used for the anatomical/molecular data fusion and statistical analyses. Regions of Interest (ROIs) were outlined and measured using tools provided by ASIPro™. Radioactivity in different brain areas was also quantified using this software. All images were displayed using the same color scale, shown in figure 2. Tracer accumulations in the ROIs in the left frontal and left temporal cortex were converted to SUVs. The SUVs for the ROIs were compared between control and treatment groups at a variety of time points using RM ANOVA. All values are presented as means+S.E.M. A p value of less than 0.05 was considered statistically significant.

Figure 2: Graph showing the dynamic uptake of 18F-FEPPA expressed as SUV vs. time for each ROI (A: left frontal cortex; B: left temporal lobe) from control, control+ALC, anesthetic-treated, anesthetic+ALC treated monkeys on PND 6/7 (n=4/group). SUV=Standard Uptake Value=average concentration of radioactivity in the ROI (MBq/mL) x body weight (gram)/injected dose (MBq). Data are shown as the means ± S.E.M. *p<0.05

Dynamic [¹⁸F]-FEPPA uptake in the cerebral cortex of animals exposed to anesthesia on PND 5 or 6

The first microPET scans of the neonatal monkey brains weretaken on PND 6/7 (one day after the exposure). The follow-up microPET scans were repeated for each monkey on PND 14 (~one week after exposure), and at 1 month (~three weeks after exposure) and 6 months of age.

For each microPET scan, images were obtained over 2 h following the injection of [¹⁸F]-FEPPA and Time Activity Curves (TACs) from the frontal and temporal cortices (the most vulnerable brain areas) [12,14] were obtained (Figure 2). Tracer accumulations in the ROIs were converted to SUVs. [¹⁸F]-FEPPA was observed in the ROIs in both control and treated animals demonstrating that it was capable of accessing these brain areas. Examples of the first microPET scans (24 hours after exposure) are shown in figure 2 as coronal, axial and sagital images highlighting [¹⁸F]-FEPPA brain concentrations in a treated monkey. The accumulation of [¹⁸F]-FEPPA was clearly increased in cortical areas such as the temporal lobe, with significant differences being observed at some time points (Figure 4). The uptake of [¹⁸F]-FEPPA in the frontal lobe of treated animals was not statistically significantly increased even though the SUVs at most time points were higher than those in controls.

In order to examine whether acetyl-l-carnitine could prevent or lessen the inhalation anesthetic–induced increase in FEPPA uptake PND 5/6 monkeys were exposed to the anesthetic combination (70% N₂O/30% oxygen and 1% ISO) for 8 h in the presence or absence of acetyl-l-carnitine (100 mg/kg, i.p.). An effect interpreted as protective was evidenced by a significant attenuation of the uptake of radiotracer in the temporal cortex byacetyl-l-carnitine.

Dynamic [¹⁸F]-FEPPA uptake in the cerebral cortex on PND 14

On PND14, approximately one week after the anesthetic exposure, SUVs in both the frontal and temporal cortical areas were higher in the exposed animals than in the controls at all time points, demonstrating an increased uptake and retention of [¹⁸F]-FEPPA in treated subjects (Figure 3). Statistically significant differences occurred only in the frontal cortical area. Again, ALC treatment resulted in a significant diminution in the uptake of radiotracer in the frontal cortex (Figure 4).

Figure 3: A set of representative microPET images (coronal plane) of a control (A) an anesthetic-treated (B), an anesthetic+ALC (C), and a control+ALC (D) treated monkey brain (B) on PND 14.

Figure 4: Graph showing the dynamic uptake of 18F-FEPPA expressed as SUV vs. time in the ROI (A: left frontal cortex; B: left temporal lobe) from control, control+ALC, anesthetic-treated, anesthetic+ALC treated monkeys on PND 14 (n=4/group). SUV=Standard Uptake Value=average concentration of radioactivity in the ROI (MBq/ mL) × body weight (gram)/injected dose (MBq). Data are shown as the means ± S.E.M. *p<0.05

Dynamic [¹⁸F]-FEPPA uptake in the cerebral cortex on PND 30 and at 6 months of age

On PND 30, about 3 weeks after anesthetic exposure, the uptake of [¹⁸F]-FEPPA in the anesthetic-exposed monkeys was higher than that in the controls at most of the time points, although no significant differences were observed at any time (Figure 5).

Figure 5: Graph showing the dynamic uptake of 18F-FEPPA expressed as SUV vs. time in the ROI (A: left frontal cortex; B: left temporal lobe) from control, control+ALC, anesthetic-treated, anesthetic+ALC treated monkeys on PND 30 (n=4/group). SUV=Standard Uptake Value= average concentration of radioactivity in the ROI (MBq/ mL) × body weight (gram)/injected dose (MBq). Data are shown as the means ± S.E.M. *p<0.05

At the age of 6 months, the uptake of [¹⁸F]-FEPPA in the anestheticexposed monkeys was similar to that of controls at most of the time points and no significant differences were observed at any time.

Peripheral benzodiazepine receptors (PBR) are mainly located on the outer mitochondrial membrane, the nuclear fractions of normal and cancerous tissue, and in the plasma membrane and membrane of organelles in various cell types [31-34]. In the CNS, PBRs are primarilylocated in glial cells, particularly microglia and astrocytes that participate in multiple physiological functions including neurosteroid synthesis, nutritional support of neurons and modulation of CNS immune reactions [35,36]. The expression of the PBR in brain is significantly increased in response to a wide variety of CNS insults. Following exposure to neurotoxicants and after other forms of neuronal insult, PBR levels increase significantly in both astrocytes and microglia in damaged brain areas in a time-dependent and regionspecific fashion. After brain injury, immunolabelling of PBRs has been shown to increase with the increase being localized to mitochondria and other cellular compartments [36-38].

Astrogliosis, an increase in the number of astrocytes generally caused by the destruction of nearby neurons, is considered a universal marker of neurotoxicity/neurodegeneration. Therefore, the PBR is widely recognized as an important target for PET imaging because levels of the PBR increase in areas of insult. Specific ligands for the PBR have been radiolabeled and used in in vivo studies in nonhuman primates. Among them, the [¹⁸F]-radiolabelled phenoxyanilide, [¹⁸F]- FEPPA can be efficiently prepared in high radiochemical yields and at high specific activity. This compound has appropriate lipophilicity for assisting brain penetration and has shown moderate uptake in the rat brain [29,39-41]. Our previous imaging study showed that prolonged exposure to a combination of N₂O and ISO during early development results in neuronal damage in the rhesus monkey [6]. To determine whether injury to brain tissue caused by anesthetic exposure-as evidenced by increases in [¹⁸F]-FEPPA uptake-can be ameliorated, the present studies were conducted to assess the efficacy of the putative neuroprotectant, acetyl-L-carnitine.

Acetyl-L-carnitine (ALC), an esterified compound of L-carnitine (LC), is essential for the β-oxidation of long chain fatty acids in mitochondria to generate ATP. As a widely distributed, short-chain ester of LC in the body, ALC is present in relatively high levels in the brain where the ratio of ALC to free carnitine is highest [42-45]. Compared to LC, ALC is much more actively transported across the blood-brain barrier and, thus, can more easily affect brain metabolism [15-18]. It has been reported that ALC provides neuroprotective benefits in neurodegenerative diseases and situations of metabolic stress such as ischemia, hypoxia, aging, alcoholism and in traumatic injury [19- 26,45-47]. Although the mechanism(s) underlying this neuroprotective effect is not known, it may involve an enhancement of mitochondrial function, its antioxidant activity, it’s abiity to stabilize membranes, use energy from the glycolytic pathway, and/or modulate the expression of genes and their proteins that exert anti-oxidant and neurotrophic effects [17,45,48,49]. It has been demonstrated in a variety of in vitro and in vivo studies that ALC can prevent neuronal death, decrease the loss of dorsal root ganglion cells, enhance the binding of nerve growth factor, and accelerate the regeneration of neurons [45,50,51].

In our previous study [12], brain damage was demonstrated in PND 7 rat pups exposed to N₂O (75%) + ISO (0.55%) with maximal neurodegeneration observed 6–8 h after exposure. This inhaled anesthetic-induced neuronal apoptosis was blocked by the co-administration of LC which was associated with changes in the expression of Bax and BCL-XL. Consistent with these previous experiments, thecurrent study demonstrated that co-treatment of ALC can attenuate the increased uptake of a PBR tracer in cerebral cortex of nonhuman primates exposed to the same inhaled anesthetics. Following such exposures during the brain growth spurt period, it seems that neuronal apoptosis can be initiated by either an intracellular factor, such as increased calcium influx and oxidative stress or by extracellular factors such as inflammatory cytokines. In a lipopolysaccharideinduced systemic inflammation model, ALC significantly decreased levels of TNFα and interleukins and consequently reduced the inflammatory reactions in the brain following acute insult [26,52,53]. This may also partly explain the reason that co-administration of ALC effectively blocked the increase in FEPPA uptake in both the temporal and frontal lobes in the exposed brain of the nonhuman primate.

ALC has been widely used in clinical trials [45] and is considered safe with a very low incidence of significant side effects during longterm administration [54]. Although multiple potential mechanisms of neuroprotection by ALC may be involved--and it is hard to accurately identify the most critical one-it seems appropriate to suggest that ALC be employed as a potential neuroprotective agent to prevent anesthetics-induced brain damage and the development of subsequent long-term neurological deficits.

This document has been reviewed in accordance with United States Food and Drug Administration (FDA) policy and approved for publication. Approval does not signify that the contents necessarily reflect the position or opinions of the FDA nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the FDA.

This work was supported by the National Center for Toxicological Research (NCTR) (NCTR E-7285)/U.S. Food and Drug Administration (FDA).