Global Journal of Anesthesiology MicroPET/CT Assessment of Minocycline Effects on Anesthetic-Induced Neuronal Injury in Developing Rats

Ketamine is a dissociative anesthetic that is frequently used for the induction and maintenance of general anesthesia in children. It has been reported that blockade of NMDA receptors by ketamine may cause neurotoxicity in neonatal rats when given over a 12 hour period during the brain growth spurt. Noninvasive, quantitative imaging of rodent brains may allow for the detection of functional, morphological and metabolic alterations induced by ketamine. Since it is known that level of the mitochondrial translocator protein (TSPO), formerly known as the peripheral benzodiazepine receptor (PBR) increase in areas of neuronal injury following exposure to neurotoxicants, TSPOs are widely recognized as important targets for imaging using positron emission tomography (PET). In this study, the effect of ketamine on the uptake and retention of [18F]-FEPPA (a TSPO ligand) in the brains of rats and the potential protective effect of minocycline, an anti-in ﬂ ammatory agent, on anesthetic-induced neuronal cell death were investigated using microPET/CT imaging. On postnatal day 7 (PND 7), rat pups in the experimental group were exposed to 6 injections of ketamine (20 mg/kg at 2 h intervals) with or without minocycline (45mg/ kg i.p. 30 minutes prior to, and 4 hours after exposure); control pups received 6 injections of saline. On PNDs 14, 21, 28 and 35, [18F]-FEPPA (18.5 MBq) was injected into the tail vein of treated and control rats and microPET/CT images were obtained over the next 90 minutes. Radiolabeled tracer accumulation in regions of interest (ROIs) in the frontal cortex was converted into Standard Uptake Values (SUVs). In PND 14 and 21 rats the uptake of [18F]-FEPPA was signi ﬁ cantly increased and the duration of tracer wash-out was prolonged in ketamine-treated rats. The increased uptake of the tracer was attenuated by the co-administration of minocycline. As expected, no signi ﬁ cant difference in radiotracer uptake in the frontal cortex was observed at 28 or 35 days after anesthesia. This preliminary study demonstrates that microPET imaging is capable of distinguishing differences in retention of [18F] -FEPPA in the brains of rodents and suggests that this approach may provide a minimally-invasive biomarker of pathogenic process associated with neurotoxicity induced by ketamine. Minocycline effectively blocks the neuronal injury caused by ketamine anesthesia in the developing rat brain.


Introduction
As a noncompetitive NMDA receptor antagonist, ketamine is a commonly used anesthetic in pediatric patients for the induction and maintenance of general anesthesia, usually in combination with other sedative and anesthetic drugs [1,2]. However, numerous studies of both the developing animal brain and primary cultured neurons have reported that prolonged exposure to ketamine during the brain growth spurt period induces widespread neurotoxicity as shown by increased neuroapoptosis, enhanced neuroinfl ammation and impaired neurogenesis [3][4][5][6][7][8][9][10][11]. General anesthesia maintained by ketamine during brain development can result in longlasting defi cits in brain function in nonhuman primates [12]. These observations have raised concerns regarding the use of ketamine-induced anesthesia for surgery or other clinical procedures in early childhood. Recently, retrospective reports have indicated that the development of cognitive abnormalities and learning disabilities in children correlate with anesthetic exposure during surgery before 4 years of age [2,10,[13][14][15][16][17][18].
Thus, practical neuroprotective strategies that can serve to protect the developing brain from ketamine or other general anesthetic-induced neuronal injury and long-lasting cognitive impairments are urgently needed.
Minocycline is a semi-synthetic, tetracycline derivative that has antibiotic activity against a broad spectrum of bacterial types [19][20][21]. In addition to its broad antimicrobial activity, minocycline effectively crosses the blood-brain barrier and has exhibited neuroprotective effects in various types of experiment models [18][19][20][21][22][23][24]. Minocycline is approved by the infections. With high bioavailability in humans, minocycline is effi ciently absorbed by the gastrointestinal tract the average half-life is around 15 h [25,26]. The mechanisms underlying the neuroprotective effect of minocycline are not clear: minocycline may exert its neuroprotection through inhibition of microglial activation, anti-infl ammatory effects, reduction of nitric oxide synthesis and/or prevention of apoptotic cell death [19][20][21][22]24,27].
In our previous studies, the administration of multiple doses of ketamine (20 mg/kg every 2 hours, 6 times, s.c.) to PND 7 rat pups, induced widespread neuronal damage in the developing brain as indicated by increased caspase-3-, silver stain-, and Fluoro-Jade C-positive cells in neocortical areas, especially in layers II and III of the frontal cortex [11]. MicroPET scans [28], indicated that anesthetic exposure signifi cantly increased the uptake of [18F]-FEPPA in the temporal and frontal lobes of exposed nonhuman primates in a time-dependent manner.
In the current study, we initiated a microPET protocol to measure [18F]-FEPPA uptake as a quantitative marker of neuroinfl ammation and, presumably, neuronal injury in the rat brain in vivo.

Drugs
Ketamine hydrochloride (Ketaset®, Fort Dodge Animal Health, Fort Dodge, IA, USA) for injection was diluted in saline.
The purity of ketamine (> 99%) was identifi ed and confi rmed via HPLC (High-performance liquid chromatography) and mass spectrometry. Minocycline hydrochloride was purchased from Sigma-Aldrich, USA) and dissolved in sterile saline.

Animals
All animal procedures were approved by the National Center assigned to control, control plus minocycline, ketamine plus saline, and ketamine plus minocycline groups (n=3-5/group, with the same rats used on PNDs 14, 21,28 and 35). Ketamine hydrochloride or saline (10μl/g) was applied subcutaneously using a 29-gauge needle. Similar to our previous studies [11], doses of ketamine (20 mg/kg/injection) or saline were given in six injections within 12 hours (2-h/interval). In order to maintain body temperature and lessen potential stressors, pups were accommodated with their dam during the 2-h injection interval [11]. Rat pups in the groups with minocycline were given i.p. injections of 45 mg/kg minocycline (100-200μl) in saline 1/2 hr before and 4 hr after the start of ketamine administration. The minocycline solution was prepared within 24h of the experiment and kept on ice between the two injections.
After exposure on PND 7, rat pups in all groups were returned to the rodent facility until microPET scanning on PNDs 14, PND 21, PND 28 and PND 35.

MicroPET scan
MicroPET images were recorded on PNDs 14, 21, 28 and 35. General anesthesia was initially induced in rat pups using 1.5-2% isofl urane gas delivered through a custom face mask: anesthesia was maintained using 1-1.5% isofl urane throughout the PET imaging procedure. For each imaging procedure, [18F]-FEPPA (18.5 MBq) was injected into the tail vein of each animal.
Following the injection, a set of serial microPET images was collected over 90 minutes (18 frames, 5 min each) to assess the uptake of the radiotracer.

MicroPET data analysis
Medical image analysis software, ASIProTM (Concorde Microsystems, Inc, Knoxville, TN) was used in the analyses for each Region of Interest (ROI). ROIs were defi ned and quantifi ed using ROI tools provided by ASIProTM. As shown in Figure 1, all images were displayed using the same color scale. Based on our histological data, the frontal cortex was the most vulnerable region to ketamine-induced neuronal cell death [11], and thus,  Curves (TACs) for the ROI in the frontal cortex [31,32], were obtained and tracer accumulation converted to SUVs. Figure 1 show 4 representative microPET images, recorded on PND 14, of the brain from a control rat (saline only), a control rat given minocycline, a ketamine-exposed rat given minocycline and a ketamine-exposed rat given saline. Images in fi gure 1 illustrate the distribution of radiotracer 0-5 minutes following the [18F]-FEPPA injection. The radioactivity accumulated in the ROI in frontal cortex of ketamine+saline treated rat was signifi cantly higher 0-5 min after injection of radiotracer compared to the radioactivity in the same ROI of brains of saline, saline + minocycline, and ketamine+minocycline treated rats.

Dynamic [18F]-FEPPA uptake in the brain at different time points
On PND14, about one week after the anesthetic exposure,

Discussion
Ketamine is commonly used to induce and maintain general anesthesia in pediatric patients [33]. Ketamine is thought to exert its anesthetic effect through blockade of NMDA receptors, resulting in inhibition of neuronal activity in CNS. Prolonged exposure to ketamine causes widespread apoptotic neurodegeneration in the developing animal brain [3,7,9,[34][35][36][37]. Previous studies in our lab demonstrated that neuronal apoptosis in several major area of developing brain can be induced by multiple ketamine injections (20 mg/kg given every 2 hours for 6 times) to PND 7 rats, especially in the frontal cortex [11,34]. Ketamine-induced neuronal apoptosis has also been shown to occur in newborn rhesus monkeys [38], in fetal NHP brains [34], and primary cultured rat neurons [6,39]. Early exposure to ketamine leads to persistent cognitive defi cits, including impaired learning and memory in rodent and nonhuman primate models [12,40].
In the present study, microPET imaging using [18F]-FEPPA was used to repeatedly detect and quantify, in the same animal, aspects of neuronal activity thought to be associated with brain cell damage caused by ketamine-induced general anesthesia during early development. As a specifi c ligand for the Translocator protein (TSPO), a biomarker of activated microglia [41][42][43], the synthesis of [18F]-FEPPA has proven effi ciently with high radiochemical yields. [18F]-FEPPA was applied successfully in previous nonhuman primate and rodent studies [29,[44][45][46][47][48][49][50]. In response to a variety of neuronal insults, glial cells are activated and the expression of TSPO in those cells, especially microglia and astrocytes, is signifi cantly upregulated. The upregulation of TSPOs is associated with several neurological and psychological disorders including multiple sclerosis, cerebral ischemia and stroke, epilepsy, brain injury, brain infection, neurodegenerative diseases and schizophrenia [43,[51][52][53][54][55][56] Although previous studies and the current data provide evidence of general anesthesia-induced developmental   Thus, practical strategies that can protect the developing brain from general anesthesia-induced neuronal injury and possibly the long-lasting cognitive impairments that may ensue are urgently needed. In addition to other agents that have been shown to be effective in ameliorating general anestheticinduced neurotoxicity such as L-carnitine, acetyl-L-carnitine, and 7-nitroindazole [32,57,58], the neuroprotective effect of minocycline was investigated in the present study.
Minocycline is an antibiotic effective against a broad spectrum of bacteria and can easily traverse the blood-brain barrier, probably because of its high lipophilicity [19][20][21].
Recently, minocycline has been reported to have other properties in addition to its antibiotic activity including neuroprotection [19][20][21][22]24,27,59]. One of the possible mechanisms underlying the neuroprotective effect of minocycline is its ability to inhibit microglial activation [21,[59][60][61]. Microglial activation in response to neuronal insults enhances the clearance of necrotic and apoptotic neurons and the survival of surrounding healthy neurons. However, microglia activation can also be toxic to the CNS due to the associated release of free radicals and infl ammatory cytokines [59,62,63]. Based on our previous microPET data showing general anesthesia related retention of FEPPA indicating microglial activation, we sought in the present study to assess the protective effects of minocycline using microglial activation as the metric.
The results presented here indicate that microPET imaging is capable of distinguishing differences in the retention of [18F]-FEPPA in the brains of rodents with a history of general anesthesia. This approach seemingly provides a minimally invasive way in which to monitor pathogenic processes associated with the developmental neurotoxicity caused by general anesthetics such as ketamine. The data presented here also suggest that minocycline can protect the neonatal brain from ketamine-induced neuronal damage, presumably-at least in part--by inhibition of microglial activation. That minocycline might be employed as a neuroprotective agent in pediatric patients and infants in need of general anesthesia should be seriously considered. However, much yet need to be described concerning the precise timing and dosing that would be appropriate for minocycline in the pediatric setting. Toward this end, further animal studies that focus on examining other neuroprotective mechanisms underlying the effects of minocycline would be helpful as would a thorough assessment of its potential adverse effects.

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