**3.2 Sepsis affects synaptic receptor and its modulator**

After sepsis, synaptic transmission seems to be affected. Although, to date, no reports are found for the direct evidence for the aberrant synaptic transmission by sepsis, sepsisinduced inflammatory cytokine alters the synaptic function. For example, interleukin-1β, which is up-regulated after sepsis, inhibits the expression of excitatory neurotransmitter receptors such as N-methyl-d-aspartate (NMDA) receptor (Coogan A & O'Connor JJ, 1997) and α-amino-3-hydoxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (Lai AY et al, 2006). Conversely, interleukin -1β enhances the expression level of inhibitory neurotransmitter receptor, gamma-aminobutyric acid (GABA) receptor (Serantes R et al, 2006). In addition, NO synthase (NOS) were up-regulated after endotoxemia (El-Mas MM et al, 2011). These neurochemicals and their receptors are critical for synaptic transmission and plasticity. These findings suggest that the sepsis-induced downstream effects such as interleukin (i.e, cytokine) are possibly associated with imbalance of synaptic transmission and its plasticity.

### **3.3 Neuochemical methods to uncover the effect of sepsis**

Our research group is challenging to tackle this issue (Imamura Y et al, 2011). To examine this, an animal model of sepsis is used. The methods are 'cecal ligation and puncture' and 'intraperitoneal injection of lipopolysaccharide (i.e., cell wall of gram negative bacteria)'. Compared to sham-operated animals (i.e., surgical operation without cecal ligation and puncture or intraperitoneally injected saline', the difference is determined by biochemical assays.

To visualize the dynamics of neurochemicals between the sham-operated mice and septic mice, our methods are 1) immunohistochemistry, 2) immunoblotting, 3) electrophysiology. These methods are well-established to examine the pathological condition by comparing with normal condition and its effect in animal models. For 1) immunohistochemistry, the

**3. Scientific techniques of neurochemistry: How to see synaptic mechanism** 

Neurochemical studies have been performed to clarify the temporal and spatial distribution of substances (i.e., proteins, enzymes, etc.) in brain. In the brain, neuronal cells which are major components of nervous system including brain, spinal cord constitute synapses. When the neurons are electrically excitable, neurotransmitters are released from the terminal of neuronal synapses (i.e., pre-synaptic terminals). On the other hand, the other neurons which receive the neurotransmitters possess receptors (i.e., post-synaptic terminals). The receptors binding to neurotransmtters activate electrically post-synaptic neurons and thereby the neuronal information is processed in the brain (i.e., synaptic transmission). Furthermore, the synaptic transmission is modulated by various chemical mediators (i.e., carbon monooxide (CO), nitric oxide (NO) etc.). These mediators are also critical to induce synaptic plasticity (i.e., plastic changes of electrical strength or efficacy in synaptic transmission). Hence, the neurotransmitters such as glutamate, acethylcholine and monoamine are major neurochemical substances, furthermore, these chemical mediators are necessary for normal synaptic transmission and synaptic plasticity. Aberrant expressions of

After sepsis, synaptic transmission seems to be affected. Although, to date, no reports are found for the direct evidence for the aberrant synaptic transmission by sepsis, sepsisinduced inflammatory cytokine alters the synaptic function. For example, interleukin-1β, which is up-regulated after sepsis, inhibits the expression of excitatory neurotransmitter receptors such as N-methyl-d-aspartate (NMDA) receptor (Coogan A & O'Connor JJ, 1997) and α-amino-3-hydoxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (Lai AY et al, 2006). Conversely, interleukin -1β enhances the expression level of inhibitory neurotransmitter receptor, gamma-aminobutyric acid (GABA) receptor (Serantes R et al, 2006). In addition, NO synthase (NOS) were up-regulated after endotoxemia (El-Mas MM et al, 2011). These neurochemicals and their receptors are critical for synaptic transmission and plasticity. These findings suggest that the sepsis-induced downstream effects such as interleukin (i.e, cytokine) are possibly associated with imbalance of synaptic transmission

Our research group is challenging to tackle this issue (Imamura Y et al, 2011). To examine this, an animal model of sepsis is used. The methods are 'cecal ligation and puncture' and 'intraperitoneal injection of lipopolysaccharide (i.e., cell wall of gram negative bacteria)'. Compared to sham-operated animals (i.e., surgical operation without cecal ligation and puncture or intraperitoneally injected saline', the difference is determined by biochemical

To visualize the dynamics of neurochemicals between the sham-operated mice and septic mice, our methods are 1) immunohistochemistry, 2) immunoblotting, 3) electrophysiology. These methods are well-established to examine the pathological condition by comparing with normal condition and its effect in animal models. For 1) immunohistochemistry, the

**3.1 General mechanism of synaptic transmission in the brain** 

these neurochemicals impede on the synaptic functions.

**3.2 Sepsis affects synaptic receptor and its modulator** 

**3.3 Neuochemical methods to uncover the effect of sepsis**

**in septic brain** 

and its plasticity.

assays.

procedure is summarized in Figure 1A. In brief, immunohistochemical staining is usually performed in following steps: 1) septic brain is fixed in 1-4% paraformaldehyde solution in

Fig. 1. Experimental procedure. A, immunohitochemistry. B, immunoblotting

Neurochemistry in the Pathophysiology of Septic Encephalopathy 155

Fig. 2. Long-term potentialtion (LTP) in the hippocampal slices. A, hippocampal slices from mouse brain. B, LTP is induced by high frequency stimulation (100Hz for 1sec x 4 times, 5

min interval).

phosphate buffered saline (PBS, pH = 7.2-7.4), 2) the brain is immersed in sucrose suspension for dehydration, embedded in optical cutting temperature compound and frozen in -80°C until use, 3) incubation with bovine serum albumin or serum solution to prevent non-specific binding of antibody which augments the background noise, 4) very thin brain slices (10 µm thick) are incubated with suspension including 1/100 -1/500 concentration (it depends on the specificity of antibody) of primary antibodies, 4) secondary antibodies which illuminate specific wavelength (usually 300-700nm) to visualize the spatial distribution of the proteins. Statistical analyses are performed between sham-operated groups and septic groups under normalization of each signal intensity and area by the number of cells (using nuclei staining).

Difference of amount of specific proteins after sepsis can be determined by immunoblotting (Figure 1B). Following suspension of tissue samples with homogenizer, the amount of total proteins in the brain mixture is measured by bradford protein assay for normalization between sample amounts. Then, the proteins are loaded with polyacrylamide gel electrophoresis. Amount of the subject protein samples transferred to PVDF membrane are determined by the specific primary antibody and the band intensities visualized by the enzyme, horse radish peroxidase, secondary antibody.

Brain function was determined by electrophysiology. Field excitatory post-synaptic potentials (fEPSPs) recordings detect population neuronal activity from recording electrode placed in extracellular neurons. Since, the method is available when neurons are arranged coordinately, it is useful for study of long-potentiation (LTP). For LTP study, our research group uses following methods: 1) making brain slices including hippocampus (300 m thick) and maintaining them in perfusing solution (2-3 ml/min) of artificial cerebrospinal solution (ACSF) saturated with mixture gas of 95% oxygen and 5% carbon dioxide, 2) the hippocampal slices are transferred to the recording chamber and placed, 3) stimulation electrode is placed on perforant path fiber and recording is performed from neurons in dentate gyrus, 4) LTP is induced by the high frequency stimulation (100 Hz for 1sec x 4times, interval 5min)(Figure 2).

As a result, we find that endotoxemia after cecal ligation and puncture results in occludin (i.e., a marker for blood brain barrier) disruption (Figure 3), increased interleukin-1β on microglial cells in the hippocampus of the brain (Figure 4) and up-regulated interleukin-1β receptor on neurons (Figure 5).

Importantly, LTP is unable to induce in a group of septic mice (Figure 6B). Several lines of studies indicate that interleukin-1β inhibits LTP (Katsuki H et al, 1990; Ross FM et al, 2003). Due to these suggestions, we apply interleukin-1β antagonist before LTP induction. Interleukin-1β antagonist effectively cancels LTP deficiency after cecal ligation and puncture (Figure. 6C). These findings strongly suggest that increased interleukin in the brain after sepsis results in synaptic plasticity deficiency. Our approach successfully demonstrates the synaptic functional deficiency in septic encephalopathy and relevance of interleukin antagonist for the deficiency. However, there are still unclear for the following points: 1) effects of other inflammatory mediators, such as high mobility group box (HMGB)-1 (Zhao X et al, 2011) and tumor necrosis factor (Clark IA et al, 2010) which are expressed abundantly in the brain, to synaptic function, 2) How is the consequence of sepsis on the other brain regions except hippocampus. In following section, relevance of autonomic nervous system and a novel technology for the treatment and prescription of septic encephalopathy in the future are discussed.

phosphate buffered saline (PBS, pH = 7.2-7.4), 2) the brain is immersed in sucrose suspension for dehydration, embedded in optical cutting temperature compound and frozen in -80°C until use, 3) incubation with bovine serum albumin or serum solution to prevent non-specific binding of antibody which augments the background noise, 4) very thin brain slices (10 µm thick) are incubated with suspension including 1/100 -1/500 concentration (it depends on the specificity of antibody) of primary antibodies, 4) secondary antibodies which illuminate specific wavelength (usually 300-700nm) to visualize the spatial distribution of the proteins. Statistical analyses are performed between sham-operated groups and septic groups under normalization of each signal intensity and area by the

Difference of amount of specific proteins after sepsis can be determined by immunoblotting (Figure 1B). Following suspension of tissue samples with homogenizer, the amount of total proteins in the brain mixture is measured by bradford protein assay for normalization between sample amounts. Then, the proteins are loaded with polyacrylamide gel electrophoresis. Amount of the subject protein samples transferred to PVDF membrane are determined by the specific primary antibody and the band intensities visualized by the

Brain function was determined by electrophysiology. Field excitatory post-synaptic potentials (fEPSPs) recordings detect population neuronal activity from recording electrode placed in extracellular neurons. Since, the method is available when neurons are arranged coordinately, it is useful for study of long-potentiation (LTP). For LTP study, our research group uses following methods: 1) making brain slices including hippocampus (300 m thick) and maintaining them in perfusing solution (2-3 ml/min) of artificial cerebrospinal solution (ACSF) saturated with mixture gas of 95% oxygen and 5% carbon dioxide, 2) the hippocampal slices are transferred to the recording chamber and placed, 3) stimulation electrode is placed on perforant path fiber and recording is performed from neurons in dentate gyrus, 4) LTP is induced by the high frequency stimulation (100 Hz for 1sec x

As a result, we find that endotoxemia after cecal ligation and puncture results in occludin (i.e., a marker for blood brain barrier) disruption (Figure 3), increased interleukin-1β on microglial cells in the hippocampus of the brain (Figure 4) and up-regulated interleukin-1β

Importantly, LTP is unable to induce in a group of septic mice (Figure 6B). Several lines of studies indicate that interleukin-1β inhibits LTP (Katsuki H et al, 1990; Ross FM et al, 2003). Due to these suggestions, we apply interleukin-1β antagonist before LTP induction. Interleukin-1β antagonist effectively cancels LTP deficiency after cecal ligation and puncture (Figure. 6C). These findings strongly suggest that increased interleukin in the brain after sepsis results in synaptic plasticity deficiency. Our approach successfully demonstrates the synaptic functional deficiency in septic encephalopathy and relevance of interleukin antagonist for the deficiency. However, there are still unclear for the following points: 1) effects of other inflammatory mediators, such as high mobility group box (HMGB)-1 (Zhao X et al, 2011) and tumor necrosis factor (Clark IA et al, 2010) which are expressed abundantly in the brain, to synaptic function, 2) How is the consequence of sepsis on the other brain regions except hippocampus. In following section, relevance of autonomic nervous system and a novel technology for the treatment and prescription of septic

number of cells (using nuclei staining).

4times, interval 5min)(Figure 2).

receptor on neurons (Figure 5).

encephalopathy in the future are discussed.

enzyme, horse radish peroxidase, secondary antibody.

Fig. 2. Long-term potentialtion (LTP) in the hippocampal slices. A, hippocampal slices from mouse brain. B, LTP is induced by high frequency stimulation (100Hz for 1sec x 4 times, 5 min interval).

Neurochemistry in the Pathophysiology of Septic Encephalopathy 157

Fig. 4. Up-regulation of interleukin (IL)-1 on Iba1-positive cells after cecal ligation and puncture (CLP). (A) Staining of IL-1β immunoreactivity (green), Iba1 (marker for microglial and/or macrophage phagocytosis) immunoreactivity (red), and DAPI (nuclei, blue) in hippocampus from sham-operated mice (left panels) and CLP-treated mice (right panels). Magnified view shown in the lower right-hand corner. Merge: white arrow heads in higher magnification indicate Iba1 positive cells in the IL-1β positive cells. Scale bar: 200 μm (lower magnification), 50 μm (higher magnification). Higher intensity of IL-1β (B) and Iba1 (C) is seen in CLP mice. (D) The ordinate shows the percentage of Iba1-positive cells in the IL-1-positive cells. The numbers of immunopositive cells were counted between slices, respectively. Almost all (>80%) IL-1-positive cells are Iba1-positive after CLP. Bar represents mean±SEM; (*n*=7:

sham, *n*=6: CLP-treated). \* *P*<0.01 (Student *t*-test). (from Imamura et. al, (2011))

Fig. 3. Blood brain barrier disruption after cecal ligation and puncture. Occludin: a marker of blood brain barrier component. (A) Occludin immunoreactivity (a marker for blood brain barrier) in hippocampus from sham-operated mice (left panel) and cecal ligation and puncture (CLP) -treated mice (right panel). Magnified view shown in the lower right-hand corner. Red: occludin, blue: DAPI. (B) Lower intensity of occludin immunoreactivity in mice after cecal ligation and puncture (CLP). In subsequent experiments, areas of hippocampus surrounded by dashed white lines were analyzed. Exposure time of UV laser for immunodetection using each marker was normalized by DAPI signal intensities. Scale bar: 200 μm (lower magnification), 50 μm (higher magnification). Bar represents mean±SEM; (*n* = 7: sham, *n* = 6: cecal ligation and puncture). \* *P* < 0.01 (Student *t*-test). (from Imamura et. al, (2011))

#### **4. Cholinergic anti-inflammatory pathway**

Sepsis affects autonomic systems (Tracey KJ, 2002). In lipopolysaccharide-injected rat (i.e., an animal model of sepsis), electrical threshold to induce action potentials are increased (i.e., hard neuronal firing) in vagus nerve (Huang J et al, 2010). This finding suggests that the vagus nerve activity is reduced after sepsis. Conversely, when the vagus nerve is electrically charged by external stimulation electrode, sepsis is alleviated (Huston JM et al, 2006; Rosas-Ballina M et al, 2008; Vida G et al, 2011). The vagus nerve projects parasympathetic neurons to all organs except adrenal gland and controls acetylcholinergic synaptic transmission. Wang and colleagues ,using 7-subunit of acetylcholine receptor knockout mice, demonstrate that vagus nerve stimulation inhibits excess release of tumor necrosis factor (i.e., activator of inflammatory mediator) in wild-type mice but not in acetylcholine knockout mice (Wang H et al, 2003). In addition, vagus nerve stimulation inhibits organ injury in the lung (Boland C et al, 2011), gut (Eisner F et al, 2011), spleen (Vida G et al, 2011) after sepsis. Furthermore, excess release of high mobility group box (HMGB)-1

Fig. 3. Blood brain barrier disruption after cecal ligation and puncture. Occludin: a marker of blood brain barrier component. (A) Occludin immunoreactivity (a marker for blood brain barrier) in hippocampus from sham-operated mice (left panel) and cecal ligation and puncture (CLP) -treated mice (right panel). Magnified view shown in the lower right-hand corner. Red: occludin, blue: DAPI. (B) Lower intensity of occludin immunoreactivity in mice after cecal ligation and puncture (CLP). In subsequent experiments, areas of hippocampus

immunodetection using each marker was normalized by DAPI signal intensities. Scale bar: 200 μm (lower magnification), 50 μm (higher magnification). Bar represents mean±SEM; (*n* = 7: sham, *n* = 6: cecal ligation and puncture). \* *P* < 0.01 (Student *t*-test). (from Imamura et. al,

Sepsis affects autonomic systems (Tracey KJ, 2002). In lipopolysaccharide-injected rat (i.e., an animal model of sepsis), electrical threshold to induce action potentials are increased (i.e., hard neuronal firing) in vagus nerve (Huang J et al, 2010). This finding suggests that the vagus nerve activity is reduced after sepsis. Conversely, when the vagus nerve is electrically charged by external stimulation electrode, sepsis is alleviated (Huston JM et al, 2006; Rosas-Ballina M et al, 2008; Vida G et al, 2011). The vagus nerve projects parasympathetic neurons to all organs except adrenal gland and controls acetylcholinergic synaptic transmission. Wang and colleagues ,using 7-subunit of acetylcholine receptor knockout mice, demonstrate that vagus nerve stimulation inhibits excess release of tumor necrosis factor (i.e., activator of inflammatory mediator) in wild-type mice but not in acetylcholine knockout mice (Wang H et al, 2003). In addition, vagus nerve stimulation inhibits organ injury in the lung (Boland C et al, 2011), gut (Eisner F et al, 2011), spleen (Vida G et al, 2011)

surrounded by dashed white lines were analyzed. Exposure time of UV laser for

after sepsis. Furthermore, excess release of high mobility group box (HMGB)-1

**4. Cholinergic anti-inflammatory pathway** 

(2011))

Fig. 4. Up-regulation of interleukin (IL)-1 on Iba1-positive cells after cecal ligation and puncture (CLP). (A) Staining of IL-1β immunoreactivity (green), Iba1 (marker for microglial and/or macrophage phagocytosis) immunoreactivity (red), and DAPI (nuclei, blue) in hippocampus from sham-operated mice (left panels) and CLP-treated mice (right panels). Magnified view shown in the lower right-hand corner. Merge: white arrow heads in higher magnification indicate Iba1 positive cells in the IL-1β positive cells. Scale bar: 200 μm (lower magnification), 50 μm (higher magnification). Higher intensity of IL-1β (B) and Iba1 (C) is seen in CLP mice. (D) The ordinate shows the percentage of Iba1-positive cells in the IL-1-positive cells. The numbers of immunopositive cells were counted between slices, respectively. Almost all (>80%) IL-1-positive cells are Iba1-positive after CLP. Bar represents mean±SEM; (*n*=7: sham, *n*=6: CLP-treated). \* *P*<0.01 (Student *t*-test). (from Imamura et. al, (2011))

Neurochemistry in the Pathophysiology of Septic Encephalopathy 159

Fig. 6. Septic encephalopathy (SE) results in long-term potentiation (LTP) deficiency by interleukin (IL)-1β. (A) Successful rates of recording of field excitatory post-synaptic potentials (fEPSP) from sham-operated mice and cecal ligation and puncture (CLP)-treated mice. Hippocampal perforant-path fibers were stimulated with test pulse stimulation (30-s intervals, 200-μs duration, half-maximum intensity). fEPSP was recorded at the dentate gyrus at a distance of 300 μm from the stimulation electrode (*P*<0.002, Student *t*-test). (B) Schematic showing LTP deficiency after CLP. LTP experiments after CLP were performed according to the schematic, which shows the time course of the experimental procedure. LTP was induced by high-frequency stimulation (100 Hz x 1 s, four times, at 5-min intervals indicated by the arrows). Representative traces of baseline at *t* = 0 min and LTP at *t* = 80 min are shown. Black trace: baseline fEPSP, red trace: fEPSP after LTP. (C) Schematic showing cancelation of LTP deficiency in CLP-treated mice by IL-1R antagonist (IL-1ra). Brain slices were incubated in the presence of IL-1ra for 30 min before LTP recording. Note that IL-1ra is effective for LTP recovery after CLP, and IL-1ra reagent has no effect on fEPSP amplitudes. Bar represents mean±SEM; (*n* = 7: sham, *n* = 7: CLP-treated). \* *p* < 0.01 (Student *t*-test). (from

Imamura et. al, (2011))

Fig. 5. Increased interleukin (IL)-1 receptor on hippocampal neurons after cecal ligation and puncture (CLP). (A) Triple staining of DAPI, type 1 IL-1 receptor (IL-1R1) and NF200. Blue: DAPI, green: IL-1R1, red: NF200. Magnified view shown in the lower right-hand corner. Merge: white arrow heads in higher magnification indicate NF200 positive cells in the IL-1R1 positive cells. Scale bar: 200 μm (lower magnification), 50 μm (higher magnification). (B) Slight decrease in neuronal fibers after CLP**.** (C) Increased IL-1R1 intensity after CLP. (D) Almost all (>90%) IL-1R1s are distributed on neurons. Bar represents mean±SEM; (*n*=7: sham, *n*=6: CLP-treated). \* *P*<0.01 (Student *t*-test). (from Imamura Y et. al, (2011))

Fig. 5. Increased interleukin (IL)-1 receptor on hippocampal neurons after cecal ligation and puncture (CLP). (A) Triple staining of DAPI, type 1 IL-1 receptor (IL-1R1) and NF200. Blue: DAPI, green: IL-1R1, red: NF200. Magnified view shown in the lower right-hand corner. Merge: white arrow heads in higher magnification indicate NF200 positive cells in the IL-1R1 positive cells. Scale bar: 200 μm (lower magnification), 50 μm (higher magnification). (B) Slight decrease in neuronal fibers after CLP**.** (C) Increased IL-1R1 intensity after CLP. (D) Almost all (>90%) IL-1R1s are distributed on neurons. Bar represents mean±SEM; (*n*=7: sham, *n*=6: CLP-treated). \* *P*<0.01 (Student *t*-test). (from Imamura Y et. al, (2011))

Fig. 6. Septic encephalopathy (SE) results in long-term potentiation (LTP) deficiency by interleukin (IL)-1β. (A) Successful rates of recording of field excitatory post-synaptic potentials (fEPSP) from sham-operated mice and cecal ligation and puncture (CLP)-treated mice. Hippocampal perforant-path fibers were stimulated with test pulse stimulation (30-s intervals, 200-μs duration, half-maximum intensity). fEPSP was recorded at the dentate gyrus at a distance of 300 μm from the stimulation electrode (*P*<0.002, Student *t*-test). (B) Schematic showing LTP deficiency after CLP. LTP experiments after CLP were performed according to the schematic, which shows the time course of the experimental procedure. LTP was induced by high-frequency stimulation (100 Hz x 1 s, four times, at 5-min intervals indicated by the arrows). Representative traces of baseline at *t* = 0 min and LTP at *t* = 80 min are shown. Black trace: baseline fEPSP, red trace: fEPSP after LTP. (C) Schematic showing cancelation of LTP deficiency in CLP-treated mice by IL-1R antagonist (IL-1ra). Brain slices were incubated in the presence of IL-1ra for 30 min before LTP recording. Note that IL-1ra is effective for LTP recovery after CLP, and IL-1ra reagent has no effect on fEPSP amplitudes. Bar represents mean±SEM; (*n* = 7: sham, *n* = 7: CLP-treated). \* *p* < 0.01 (Student *t*-test). (from Imamura et. al, (2011))

Neurochemistry in the Pathophysiology of Septic Encephalopathy 161

Fig. 7. Lymph node imaging by near infra-red q-dot (from Jin T. (2008))

encephalopathy in higher temporal resolution in the future studies.

**6. Conclusion** 

Near infrared spectroscopy (NIRS) uses near infra-red region (800-2500nm) of light. Since the light in near infra -red range can pass through the skull, reach to the surface of the brain and coincide with the fast event-related responses in human recorded by electroencephalograms (Medvedev AV et al, 2010), it has been used to track cerebral hemodynamics (Gratton E et al, 2005) and human brain activity(Perrey S, 2008). On the other hand, trials to apply q-dots to the molecules associated with pathological states are performed in neurosurgery (Taghva A et al, 2010) and brain tumor (Popescu MA & Toms SA, 2006). Therefore, if labeling of brain with q-dot which emits the fluorescence light in the near infra-red region becomes successful, we can track the molecules associated with septic

Septic encephalopathy is associated with many kinds of inflammatory cytokines, receptors, chemicals such as interleukin family, toll-like receptor, tumor necrosis factor, high mobility group box. Since these inflammatory molecules induce systemic inflammatory response in the whole body and affect the brain function after blood brain barrier disruption, they occurs neurological symptoms such as vasogenic edema, aberrant synaptic transmission and plasticity. In addition, the weakness of vagus nerve after septic encephalopathy is related to the pathophysiology. Although the molecular mechanisms for sepsis are explained, medical

(i.e., inflammatory mediator) from macrophage is also inhibited by vagus nerve stimulation (Wang H et al, 2004). The neural mechanism of vagus nerve stimulation is summarized in review (Tracey KJ, 2009). The review describes the mechanisms as follows: when sepsis is induced by endotoxemia, a site of infection imposes cytokine release through NF-kappaB (nuclear factor kappa-light-chain enhancer of activated B cells) activation. This signaling pathway accelerates cytokine production and thereby cell death via cytokine storm. When the vagus nerve from the brain stem is stimulated, it activates cholinergic anti-inflammatory pathway (efferent arc) and inhibits innate immune response in the spleen via activation of nicotinic acetylcholine receptor. The nicotinic acetylcholine receptor activation suppresses NF-kappaB activation via JAK2 (Janus kinase 2, cytokine receptor) - STAT3 (signal transducer and activator of transcription 3, transcription activator) pathway and the cytokine release.

Hence, vagus nerve stimulation may employ a high potential for the therapeutics of the clinical treatment of sepsis-related systemic inflammatory symptoms of septic encephalopathy.

#### **5. Future prospect: A novel technology for tracking one molecule imaging in living organism**

The findings we introduce are mainly resulted from neurochemical research assays using dissected tissues and cells. Recently, non-invasive methods such as functional magnetic resonance imaging (fMRI) are utilized to examine the brain dysfunction in living organism (rodents and human, etc). For example, in a mouse model of sepsis, accumulation of effusion by vasogenic edema is observed using fMRI in hippocampus and cortex (Bozza FA et al, 2010). Thus, fMRI is a powerful tool to find the damaged region in the brain because of the high spatial resolution. However, since fMRI detects the alteration of blood stream and estimates this as brain functional alteration, there is a limitation of the fMRI study. For example, it is hard to distinguish excitatory or inhibitory neurotransmission without a tag of each. In addition, because action potentials propagation between synapses in the brain is conducted within 100 msec (Wu JY et al, 2008), it is hard to track the temporal alteration of fast synaptic transmission with the time resolution (later than 1s) of fMRI (Tsao J, 2010). To reinforce these weak points, we address a novel nano-technology, '**one molecule imaging with quantum dot**' for the future research of septic encephalopathy.

A quantum dot (q-dot) is a nanometer-sized semiconductor particle (2-10 nm diameter). Cadmium selenide (CdSe) and cadmium telluride (CdTe) are well-utilized for the nanomaterials of q-dot. When q-dot is placed in the crystallized structure, its electrical state for the fluorescence emission is discretized (Reed MA et al, 1988). As a result, only the fluorescence with a specific wavelength is amplified. Immunohistochemical studies using qdot have a high signal to noise ratio and few photobleaching(Chan WC, Nie S, 1998). Although normal q-dots are hydrophobic, it can be changed their property to hydrophilic in order to apply to living organism. Jin T et. al (2008) develop a water-soluble glutathionecoated Q-dots. They successfully detect q-dot signals in the lymph node in the mouse (Fig. 7). In addition, q-dot is available to track the molecules expressed in the brain. Intraperitoneally-injected q-dots can be found in the brain (Kato S et al, 2010; Yeh TK et al, 2011). Hence, q-dot is a hopeful nano-material to track a molecule associated with sepsis. Then, how do we track the molecules associated with septic encephalopathy with q-dots?

(i.e., inflammatory mediator) from macrophage is also inhibited by vagus nerve stimulation (Wang H et al, 2004). The neural mechanism of vagus nerve stimulation is summarized in review (Tracey KJ, 2009). The review describes the mechanisms as follows: when sepsis is induced by endotoxemia, a site of infection imposes cytokine release through NF-kappaB (nuclear factor kappa-light-chain enhancer of activated B cells) activation. This signaling pathway accelerates cytokine production and thereby cell death via cytokine storm. When the vagus nerve from the brain stem is stimulated, it activates cholinergic anti-inflammatory pathway (efferent arc) and inhibits innate immune response in the spleen via activation of nicotinic acetylcholine receptor. The nicotinic acetylcholine receptor activation suppresses NF-kappaB activation via JAK2 (Janus kinase 2, cytokine receptor) - STAT3 (signal transducer and activator of transcription 3, transcription activator) pathway and the

Hence, vagus nerve stimulation may employ a high potential for the therapeutics of the clinical treatment of sepsis-related systemic inflammatory symptoms of septic

**5. Future prospect: A novel technology for tracking one molecule imaging in** 

The findings we introduce are mainly resulted from neurochemical research assays using dissected tissues and cells. Recently, non-invasive methods such as functional magnetic resonance imaging (fMRI) are utilized to examine the brain dysfunction in living organism (rodents and human, etc). For example, in a mouse model of sepsis, accumulation of effusion by vasogenic edema is observed using fMRI in hippocampus and cortex (Bozza FA et al, 2010). Thus, fMRI is a powerful tool to find the damaged region in the brain because of the high spatial resolution. However, since fMRI detects the alteration of blood stream and estimates this as brain functional alteration, there is a limitation of the fMRI study. For example, it is hard to distinguish excitatory or inhibitory neurotransmission without a tag of each. In addition, because action potentials propagation between synapses in the brain is conducted within 100 msec (Wu JY et al, 2008), it is hard to track the temporal alteration of fast synaptic transmission with the time resolution (later than 1s) of fMRI (Tsao J, 2010). To reinforce these weak points, we address a novel nano-technology, '**one molecule imaging** 

A quantum dot (q-dot) is a nanometer-sized semiconductor particle (2-10 nm diameter). Cadmium selenide (CdSe) and cadmium telluride (CdTe) are well-utilized for the nanomaterials of q-dot. When q-dot is placed in the crystallized structure, its electrical state for the fluorescence emission is discretized (Reed MA et al, 1988). As a result, only the fluorescence with a specific wavelength is amplified. Immunohistochemical studies using qdot have a high signal to noise ratio and few photobleaching(Chan WC, Nie S, 1998). Although normal q-dots are hydrophobic, it can be changed their property to hydrophilic in order to apply to living organism. Jin T et. al (2008) develop a water-soluble glutathionecoated Q-dots. They successfully detect q-dot signals in the lymph node in the mouse (Fig. 7). In addition, q-dot is available to track the molecules expressed in the brain. Intraperitoneally-injected q-dots can be found in the brain (Kato S et al, 2010; Yeh TK et al, 2011). Hence, q-dot is a hopeful nano-material to track a molecule associated with sepsis. Then, how do we track the molecules associated with septic encephalopathy with q-dots?

**with quantum dot**' for the future research of septic encephalopathy.

cytokine release.

encephalopathy.

**living organism** 

Fig. 7. Lymph node imaging by near infra-red q-dot (from Jin T. (2008))

Near infrared spectroscopy (NIRS) uses near infra-red region (800-2500nm) of light. Since the light in near infra -red range can pass through the skull, reach to the surface of the brain and coincide with the fast event-related responses in human recorded by electroencephalograms (Medvedev AV et al, 2010), it has been used to track cerebral hemodynamics (Gratton E et al, 2005) and human brain activity(Perrey S, 2008). On the other hand, trials to apply q-dots to the molecules associated with pathological states are performed in neurosurgery (Taghva A et al, 2010) and brain tumor (Popescu MA & Toms SA, 2006). Therefore, if labeling of brain with q-dot which emits the fluorescence light in the near infra-red region becomes successful, we can track the molecules associated with septic encephalopathy in higher temporal resolution in the future studies.
