An increased HDC cell count in the tuberomammillary
nucleus in Narcolepsy type 1 patients
https://www.chionfoundation.org/single-post/2017/06/28/What-is-Narcolepsy
Desirée van Dam (11688785)
Bovenkarspel, 1-07-2020
Netherlands institute for Neurosciences
Swaab group
An increased HDC cell count in the tuberomammillary
nucleus in Narcolepsy type 1 patients
Desirée van Dam (11688785)
Total word count: 4537 words
Bovenkarspel, 1-07-20
Universiteit van Amsterdam
Science Park 904, 1098 XH Amsterdam
Faculty of Science
Bachelor Biomedical Sciences
2
ndsupervisor: Prof. dr. Helmut Kessels
Netherlands institute for Neuroscience
Meibergdreef 47, 1105 BA Amsterdam
Swaab Group
Table of contents
1. Abstract……….4
2. Introduction………5
3. Materials & methods………...7
3.1 Human brain material………7
3.2 H3R immunocytochemical staining………..7
3.2.1 Pilots………7
3.2.2 Optimized protocol………..7
3.3 HDC immunocytochemical staining……….9
3.3.1 Human brain material……….9
3.3.2 HDC staining protocol……….9
3.4 Statistic analysis……… 10
4. Results……….11
4.1 Increase in HDC in Narcolepsy type 1 patients……….………11
4.2 Negative correlation between orexin neurons and HDC cells in Narcolepsy type 1 patients … ……….11
5. Discussion……… 12
6. Reference list………..15
Appendix I. Protocol for H3R immunocytochemical staining……….18
Appendix II. Protocol for HDC immunocytochemical staining ………..20
Abstract
Narcolepsy is a sleep/wake disorder, where patients show excessive daytime sleepiness among other symptoms, some patients show cataplexy, a sudden loss of muscle tone (Narcolepsy type 1).
Narcolepsy is caused by the degeneration of orexin/hypocretin. The Tuberomammillary nucleus, where histamine, is produced, plays a key role in maintaining wakefulness. Histidine decarboxylase is the key enzyme to produce neuronal histamine. Neuronal Histamine are regulated by Histamine-3 receptor.
It is however not known if there is a changed expression of Histidine decarboxylase or Histamine-3 receptor in Narcolepsy patients.
This study looked at HDC expression in Narcolepsy type 1 patients by immunocytochemical staining. The results have shown an increase in HDC cells in Narcolepsy type 1 patients. H3R deserves future study as this is a therapeutic target for excessive day time sleepiness of narcolepsy.
2. Introduction
Narcolepsy is a sleep/wake disorder, in which patients show excessive daytime sleepiness (EDS), short rapid-eye movement (REM) sleep onset, sleep paralysis, hypnagogic hallucinations, some patients also show cataplexy (sudden loss of muscle tone, called narcolepsy type 1) (Dauvilliers et al., 2007). A characteristic of narcolepsy type 1 is a nearly complete loss of orexin (also called hypocretin) neurons.
The excessive daytime sleepiness in Narcolepsy patients is caused by a disrupted regulation in the Tuberomammillary nucleus (TMN) (Shan et al., 2015). The TMN in the hypothalamus is the only source of neuronal histamine synthesis in the adult mammalian brain. (Shan et al., 2015a; Haas et al., 2008; Panula & Nuutinen, 2013). Histidine decarboxylase (HDC) is the enzyme that is involved in the histamine synthesis, HDC transfers L-histidine to histamine in histaminergic neurons, which are in the TMN (Kukko-Lukjanov & Panula, 2003). It was shown by Takahashi et al. (2006) that there is an increase in histaminergic TMN neurons during wakeful state, another study showed an increase in histamine concentration during wakefulness (Strecker et al., 2002). To add to these results Prell et al. (1989) concluded an increase in histamine metabolite during daytime in the cerebrospinal fluid (CSF). All of these results led to the conclusion that the activity of the TMN is increased during wakefulness and that it is probably the histamine that is important in this activity. Other previous studies found that histamine plays an important part in maintaining wakefulness, when histamine is inhibited the amount of rapid-eye movement sleep is increased, which leads to a disruption in sleep (Jewett, 1968; Risberg et al., 1975; Wauquier et al., 1981). Another study found similar results, when histamine is administered an increase of wakefulness was shown (Kalivas, 1982). These results led to the conclusion that histamine plays an important role in maintaining wakefulness. Histamine is thus produced in the TMN, and then transferred to vesicles by vesicular monoamine transporter 2 (VMAT2) (Merickel & Edwards, 1995) for release in the synapse. Here histamine can bind to 4 G-protein coupled receptors (GPCRs), histamine-1 receptor (H1R), H2R, H3R and H4R. H1R is involved in cellular migration, vasodilation and bronchoconstriction, H2R alters gastric acid secretion and the production of airway mucus. H3R is important in neuro-inflammatory diseases and H4R is involved in allergy and inflammation (Thangam et al., 2018). For narcolepsy the H3R is important, because narcolepsy is a neuro-inflammatory disease. In narcolepsy the orexin neurons are damaged as the result of an inflammatory response caused by histamine. Pedotti et al. (2003) found that histamine activates immunological cells and Silver et al. (1996) found that histamine increases the blood-brain barrier permeability, these actions lead to an inflammatory response at the location of histamine release. Orexin is located at the histamine release site in the TMN, due to the reciprocal connection between histamine and orexin, orexin neurons will be damaged as the result of this inflammation. Eriksson et al. (2001) concluded that histamine and orexin are reciprocally connected. Eriksson et al. (2001) found with electrophysiology that when orexin is released the firing rate of the histaminergic neurons in the TMN rises (after application of orexin, the TMN firing rate increased). They also found with immunocytochemistry that orexin neurons were heavily innervated with histaminergic axons (the two cells had overlapping regions) in the TMN.
To get back to the Histamine-3 receptor and it’s function. The Histamine-3 receptor (H3R) is an autoreceptor that inhibits the histamine synthesis and release in histaminergic neurons. When histamine binds H3R the histamine concentration in the synapse will thus decrease. But when an antagonist or inverse agonist binds the H3R the synthesis and release of histamine won’t be inhibited anymore. (Passani & Blandina, 2011). Since 2019 a new medicine (Pitolisant) for treatment of
excessive daytime sleepiness in narcolepsy type 1 and 2 was approved by the European Medicines agency (EMA) and United States Food and Drug administration (FDA). Pitolisant is a Histamine-3
receptor inverse agonist that reduces the excessive daytime sleepiness in patients with narcolepsy. (Lin, et al., 2008; Dauvilliers, et al., 2013). Binding of Pitolisant leads to an inhibition of the H3R, which leads to an increase in histamine production and histamine release. This increase leads to an increase in wakefulness and a reduction in excessive daytime sleepiness (Thorpy & Bogan, 2020). The reduction in excessive daytime sleepiness can be explained by a previous research that showed that histidine decarboxylase (HDC) -/- mice showed impaired wakefulness. (Parmentier et al., 2002). So it is known that Pitolisant is a H3R inverse agonist and that this leads to an increase in histamine concentration in the TMN, which reduces excessive daytime sleepiness in narcolepsy patients. It is however not known if Pitolisant has a positive effect on excessive daytime sleepiness due to a difference in H3R density or HDC cell count in narcolepsy patients. In this research the H3R density in Narcolepsy patients will be compared to the H3R density in healthy patients, to find out if Pitolisant reduces the excessive daytime sleepiness due to a difference in H3R density. The prediction of this research is that Narcolepsy patients have an increased H3R concentration. Narcolepsy patients would have a higher H3R concentration because they show excessive daytime sleepiness, which is caused by a lower level of histamine (Parmentier et al., 2002). When there are more H3R’s, more histamine release and synthesis can be inhibited. It will be less logical to predict that more histamine is
produced in Narcolepsy due to the fact that a receptor can only bind a maximum amount of ligand. If the excessive daytime sleepiness is caused by an increase in H3R, a reduction in histamine
concentration in the synapse would be the result, with an increase in excessive daytime sleepiness as consequence. In this research H3R will be examined in Narcolepsy post mortem brains, this will be conducted by immunocytochemical staining of the H3R in the region of the hypothalamus where the TMN cells are located. The expectation is to find an increase in H3R counts compared to healthy patients.
3. Material & methods
3.1 Human brain material
The brain material was received from the Netherlands Brain Bank (NBB) with permission from the patient or their next of kin for brain autopsy and for the use of the brain material for research purposes. The hypothalami were fixed in 0.1M phosphate buffer with 4% formaldehyde (pH 7.2) for 1-2 months. After this fixation the tissue was dehydrated in graded ethanol and embed in paraffin and cut in sections of 6 µm on a microtome. The sections were then stored in wooden boxes at room temperature (RT) until they are used. 10 brain samples were studied (Table 1), 5 samples from narcolepsy patients and 5 samples that were the control samples. The samples are matched on age, sex, post mortem delay (PMD) and cerebrospinal fluid pH.
3.2 H3R immunocytochemical staining 3.2.1 Pilots
The pilot that was conducted first was a pilot where different pH levels for the antigen retrieval were tested, to find the pH which would lead to the best antigen retrieval. Better antigen retrieval leads to a more optimal antibody binding to H3R. Antigen retrieval in pH7.6 (TBS) showed the best staining compared to the other pH levels (pH4.0, pH6.0, pH9.0), but still showed some aspecific staining (Table 2). In the second pilot different concentrations of the primary antibody were tested (1:500, 1:1000, 1:4000, 1:8000), to check which concentration would give the most ideal signal/noisy ratio of staining. All of the concentrations showed the signal/noisy ratio were too low, so another pilot with higher primary antibody concentrations (1:500, 1:200, 1:100) was tested. 1:200 gave the most ideal signal/noise ratio of staining for H3R, there was still some background (Table 3), to reduce this, SUMI-milk was employed. Different SUMI-milk percentages (1%, 2%, 5%) were used for the
incubation with primary antibody. Results of this pilot showed less background staining with the 5% SUMI-milk (Table 4). So in the final protocol TBS (pH7.6) is used for antigen retrieval, primary antibody is used in 1:200 and incubation of the primary antibody is done in SUMI-5% milk. These adjustments to the original protocol are made to get the most specific binding and reduce the background. For the protocol that was used, see Appendix I.
3.2.2 Optimized protocol
Sections were mounted in RNase free MQ and put on a slide which was placed on a hot plate at 40°C for at least one day. The slides were then deparaffinized and rehydrated in graded ethanol’s. After dehydration the slides were washed in 1x TBS, this is made from 10mL 10x TBS (450g NaO, 300g Tris, pH7.6) diluted with 90mL distilled water. For antigen retrieval slides were put in a microwave in the 1x TBS (pH7.6) for 2x 2 min at 800W as pre heating, followed by 2x 5 min at 800W. After cooling down for 30 minutes at room temperature and washing in 1x TBS slides were incubated with 180 µL (per slide) of primary antibody anti-H3R, ab124732 (abcam), 1:200 diluted in 5% SUMI-milk, for 1h. To make the SUMI, the total amount was first calculated by multiplying the amount of slides by 180 (180 µL per slide), this led to the amount of 1x TBS needed. 0.25% of the TBS volume was the weight of the gelatin that was used. Triton was added as 0.5% of the TBS volume. pH was adjusted until pH7.6. To make the 5% SUMI-milk, 5% of the total SUMI amount was added in milk powder (Campina elk) weight. pH was again adjusted until pH7.6. After the incubation in primary antibody in 5% SUMI-milk, slides were put in a 4°C room for overnight incubation. In the following day the slides were washed in 1x TBS for 3x 10 min. After washing slides were incubated with a minimal of 180 µL biotinylated secondary antibody (horse anti-rabbit, BA-1100) per section, which was dissolved in 1:400 in SUMI. The amount of SUMI needed was calculated by multiplying 180 by the amount of
slides. ABC complex (2-01-2020, vectastain ABC kit) was prepared for diluting A and B in 1:800 in SUMI, this was left on the rocking table for at least 30 min to mix. Total amount of SUMI was calculated as previously, amount of slides x 180. After incubation with the secondary antibody the slides were washed 3x 10 min in 1x TBS. Slides were then incubated with 180 µL in ABC complex in 1:800 for 1h. After the incubation, slides were washed 3x 10 min in 1x TBS before incubation with the DAB-Ni-solution was started. DAB-Ni solution was made in the proportions of 13 mL TBS, 1 mL DAB 1 mL Ni (Nickel Ammonium Sulphate, 3.45% in TBS) and 5 mL of H2O2. When everything was mixed together the solution was filtered and 180 µL was used per slide for 10 min. The reaction was stopped in 1x TBS. To finish the staining the slides were dehydrated in graded ethanol’s for 5 min in each ethanol and cleared in xylene for 2x 10 min. The slides were then covered in Entellan and a coverslip and dried overnight for further examination the next day.
NBB Age Sex PMD pH Cause of death Narcolepsy
2008-023 66 f 420 6,57 Heart failure
2010-064 85 f 220 6,77 Chronic pain syndrome with palliative sedation 2018-018 82 f 330 6,48 Pneumonia
2018-058 87 m 290 6,3 Metastatic cancer with unknown primary tumor 2018-091 71 f 395 6,32 Heart failure, renal insufficiency
Controls
2012-052 64 f 340 6,35 Palliative sedation 2000-022 83 f 470 6,52 Heart failure, Cachexia 2012-005 84 f 336 6,68 Heart failure
2009-001 88 m 283 6,17 Gastro-intestinal bleeding 1998-104 74 f 445 6,95 Necrosis of the intestines
Table 1. Information about the patients that donated the brain material.
Shown here is the information from the patients that were examined in this study, the Narcolepsy and Control group information are given. NBB: the Netherlands Brain Bank number; PMD: post-mortem delay (in minutes); pH: cerebrospinal fluid pH.
Table 2. Results from the pilot for finding the best antigen retrieval condition.
Here the different pH levels (pH 4.0, 6.0, 7.6 and 9.0) are put next to each other and their results on the staining on the human brain tissue. The best staining was obtained with pH7.6.
Antigen retrieval conditions Results
pH4.0 (Sod.Citr.) Too little staining
pH6.0 (Sod.Citr.) Aspecific staining and light staining
pH7.6 (TBS, micowave) Less aspecific staining and light stained as in the other conditions
pH7.6 (TBS, untreated) Aspecific staining and light staining pH9.0 (Tris-HCL) Aspecific staining and light staining
Table 3. Results from pilots for finding the best concentration for primary antibody to be used.
Here the different concentrations of the primary antibody are put next to each other with their results on the staining of the human brain tissue. The best staining was obtained with a primary antibody concentration of 1:200.
Table 4. Results from pilot for finding the best SUMI-milk percentage to be used.
Here the different SUMI-milk percentages (1%, 2% and 5%) are put next to each other with their results on the staining of the human brain tissue. The best staining was obtained with 5% SUMI-milk.
3.3 HDC immunocytochemical staining
Due to the corona virus circumstances the follow up experiment for the H3R staining could not be conducted. Another experiment that stained HDC in Narcolepsy patients could be conducted and analyzed. HDC is the enzyme that transfers histidine to histamine, as mentioned in the introduction. The experiment for HDC staining was conducted by dr. Ling Shan and the data that is going to be used in this report has been obtained from this research.
3.3.1 Human brain material
From the human brain material available (see 2.1) only the patients with Narcolepsy type 1 were used in this experiment.
3.3.2 HDC staining protocol
Sections were prepared the same as mentioned before (see 2.2.2), after deparaffinization and rehydration the slides were washed in aqua dest. For antigen retrieval the slides were microwave treated in 0.01M sodium citrate (pH 6.0) for 1x 2 min at 900W as pre heating, followed by 2x 5 min at 900W. The 0.01M sodium citrate solution was made with 25mL of 0.1M sodium citrate (pH 6.0) and diluted with 225mL of aqua dest. After cooling down for 30 minutes at room temperature the slides were washed in 1x TBS for 2x 10 min. The 1x TBS was prepared the same as mentioned in 2.2.2. After washing the slides were pre-incubated for 1h at room temperature with 180µL 5% TBS-milk per slide. The 5% TBS-milk was made by multiplying 180 with the amount of slides for the total amount of 1x TBS. 5% of the total TBS volume was added in milk powder (Campina elk) weight. Slides were washed 2x 10 min in 1x TBS before overnight incubation with anti-HDC (EUD2601, Acris). The anti-HDC was dissolved in 5% SUMI-milk (for preparation see 2.2.2) in 1:4000, every slide was incubated overnight in a 4 °C room with 180µL from this solution. The following steps are the same as in 2.2.2, the only difference was that the DAB-Ni-solution was left on the slides for 7 minutes instead of 10 minutes. The reaction was stopped in 1x TBS. To finish the staining the slides were dehydrated in graded
Concentrations primary antibody Results
1:100 More aspecific staining
1:200 Less aspecific staining and clear enough for
counting
1:500 More stained, but not clear enough for counting
1:1000 Lightly stained
1:4000 No staining
1:8000 No staining
SUMI-milk percentages Results
1% Glia cell staining
2% Glia cell staining
ethanol’s for 5 min in each ethanol and cleared in xylene for 2x 10 min. The slides were then covered in Entellan and a coverslip and dried overnight for further examination the next day. The stained spots were counted when there was a visible stained nucleolus present from the histaminergic cell. See Appendix II for the protocol that was used.
3.4 Statistic analysis
For the comparing of the cell counts per patient a Mann-Whitney-U test was performed. Spearman’s correlation coefficient was used for testing correlations. In both tests a p-value <0.05 was seen as significant, both tests were two-tailed.
Due to COVID-19, I am not able to get results for H3R staining between controls and
narcolepsy cases. Following results were obtained by Dr. Ling Shan for his HDC
study.
4. Results
4.1 Increase in HDC in the number of HDC positive cells in Narcolepsy type 1 patients compared to healthy patients.
Narcolepsy type 1 patients showed significantly more HDC cells compared to the healthy patients (Figure 1. Mann-Whitney-U test, Z = -2.334 and p = 0.024). The median and standard error of the mean (SEM) of the 2 groups were 74147 ± 2521 for n=3 and 50944 ± 8375 for n = 5.
Figure 1. HDC cell count in Narcolepsy type 1 patients compared to healthy patients.
Shown here are the medians ± SEM (74147 ± 2521 and 50944 ± 8375)for HDC cell count in Narcolepsy type 1 patients and healthy patients. With on the y-axis the HDC cell count.
4.2 Negative correlation between orexin neurons and HDC cells in Narcolepsy type 1 patients
In a previous research conducted by dr. Ling Shan the orexin neurons in Narcolepsy and healthy patients were counted. Narcolepsy type 1 patients had significantly less orexin neurons compared to the healthy patients (figure 3, Mann-Whitney-U test; Z = -2.324, p = 0.024). The median and SEM of the 2 groups were 4175 ± 2639 for n = 3 (Narcolepsy type 1 group) and 40492 ± 4945 for n = 6 (healthy patients). Results from this previous research and the HDC research were combined to test for a correlation. The amount of HDC cells in Narcolepsy patients were negatively correlated with orexin cell count (Spearman’s correlation coefficient; r = -0.661, p = 0.038).
Figure 3. Orexin neuron count of Narcolepsy type 1 patients compared to healthy patients.
Shown here are the medians and their SEM (4175 ± 2639 and 40492 ± 4945) for orexin neurons in Narcolepsy type 1 patients and healthy patients. With on the y-axis the amount of orexin neurons.
5. Discussion
Due to COVID-19, I am not able to get results for H3R staining between controls and
narcolepsy cases. I am trying to discuss a bit of results from Dr. Ling Shan about HDC.
The results from the HDC study have shown that Narcolepsy type 1 patients have an increased number of HDC cell numbers compared to controls. The data shows a reduction of orexin neurons in Narcolepsy type 1 patients compared to healthy patients and a negative correlation between HDC and orexin neurons in Narcolepsy type 1 patients. From these results it can be concluded that Pitolisant has a positive effect on EDS in Narcolepsy type 1 patients because Narcolepsy type 1 patients have an increased HDC cell count.The increase in HDC cell numbers in Narcolepsy type 1 patients can be explained as a result of the inflammatory response that occurs in Narcolepsy (Pedotti et al., 2003; Silver et al., 1996). With an increase in HDC, histamine level in the vesicles should also increases. Histamine level should increase because HDC is the enzyme that is involved in the histamine synthesis, HDC transfers L-histidine to
histamine in histaminergic neurons, which are in the TMN (Kukko-Lukjanov & Panula, 2003 ; Haas et
al., 2008). Histamine can bind to the H3R, when H3R is activated by histamine binding, inhibition of
the histamine synthesis and release is the result (Torrent et al., 2005). With more HDC, more histamine is available, histamine binds however to H3R, which leads to an inhibition of histamine synthesis and release. To conclude this, it can be stated that as the result of an increased HDC cell count more histamine is available, but histamine binds to H3R (which inhibits histamine) so there is less histamine available in the synapse at the end. The results from Ohtsu et al. (2001) found that HDC knock-out mice lacked histamine, which is in agreement with the previously mentioned studies about HDC, that HDC is important for histamine production (Kukko-Lukjanov & Panula, 2003 ; Haas et
al., 2008). Pitolisant, however, is a H3R inverse agonist, when Pitolisant binds to H3R the inhibition is
lifted (Lin et al., 2008; Dauvilliers et al., 2013). When the inhibition is lifted histamine synthesis and release will not be inhibited anymore, with an increase in histamine concentration in the synapse as result. An increase in the histamine level leads to an increase in wakefulness (Takahashi et al.,2006; Strecker et al., 2002) and thus reduces the EDS. To conclude this, Pitolisant reduces the amount of EDS because it lifts the effect of the increased HDC cell count which led to an decreased histamine concentration in the synapse.
The results do not align with the hypothesis that Narcolepsy patients have a higher density of H3R, H3R was not examined due to COVID-19. Based on the results of this research it can be suggested that H3R will not differ in Narcolepsy type 1 patients because there is already more HDC, which means more histamine can be released and bound to H3R. When the H3R’s are all bound the histamine production and release will be inhibited. There is however already more HDC present, all H3R’s will already be bound so there is no need for more H3R’s on the synapse to inhibit histamine. The amount of HDC cells that were counted could maybe be counted wrongly, because the same histamine cell can be present in multiple brain slices, that are sliced from frontal to dorsal. This means that it is possible to count the same HDC colored spot two or more times when a different brain slice is being examined under the microscope. The higher count of HDC cells in Narcolepsy patients could be explained due to this artifact. This was however taken into account while counting the HDC cells under the microscope. HDC cells were only counted if there was a visible nucleolus from the histaminergic cell present in the same brain slice. The chance of double counting HDC cells is reduced to zero with this adjustment. Another aspect that could lead to a false counting is the antibody that could have bound to another molecule that is not HDC, this would lead to a false counting of the amount of HDC. The antibody however has been tested by the company for the
specificity (Progen shop, anti-HDC) and it is not likely that the antibody would bind aspecifically. Besides this, the conditions in the pilots were adjusted until the best specific staining, most ideal signal/noise ratio and less background were achieved. The small amount of patients and controls that were used could have affected the conclusion, because such a small sample group does not represent the whole population. These patients can be patients that have for example the lowest HDC cell count, while other Narcolepsy type 1 patients may have a higher HDC cell count, or maybe other patients do not show a significant difference. To get results that give a better overview of the whole Narcolepsy type 1 population, at least 10 patients for example can give more reliable results. The results of this study confirmed previous studies (Peyron et al., 2000 ; Thannickal et al., 2000) where it was concluded that orexin neurons are reduced in Narcolepsy type 1 patients. Histaminergic neurons innervate the orexin neurons (Eriksson et al., 2001), which means that the inflammatory reaction can also occur here. Orexin neurons are sensitive for autoinflammatory insult (Shan et al., 2015b). An explanation for the big loss of orexin neurons in Narcolepsy type 1 patients (Peyron et al., 2000 ; Thannickal et al., 2000) can be the increased HDC cell count that was found in this study. An increase in HDC means that more histamine will be produced (Kukko-Lukjanov & Panula, 2003; Haas
et al., 2008), if there is more histamine the inflammatory reaction caused by histamine (Pedotti et al.,
2003; Silver et al., 1996; Chikahisa et al., 2013) will increase and this will destroy more orexin neurons. So the big loss of orexin neurons can then be explained by the increase in HDC in Narcolepsy type 1 patients.
The results of this study show that there is an upregulation of HDC cells in Narcolepsy type 1
patients, as a result of this upregulation there is a reduction in histamine in the synapse which leads to an increase in EDS. With the use of Pitolisant the histamine concentration will increase and patients will show a reduction in EDS. Pitolisant reduces the EDS in Narcolepsy type 1 patients by cancelling out the effect of the increased HDC concentration. With Pitolisant the socioeconomic burden for Narcolepsy patients will reduce, because their EDS is decreased. This decrease means that they will not be held back in life as much as before they took the medicine.
To find out if the orexin neurons are decreased due to an inflammatory reaction (Pedotti et al., 2003; Silver et al., 1996), a follow up study should be conducted where the brains are studied for an inflammatory reaction at the orexin neurons in the TMN for example. To know if the negative correlation between orexin and HDC cell count is the result of an inflammatory response, further research will need to be conducted. For example brains with higher HDC cell count and lower HDC cell count can be compared with their orexin cell count. To find out if it is only the HDC cell count that differs in Narcolepsy type 1 patients, the H3R concentration can be examined in a follow up study to see if this differs. This can be carried out by following the protocol in Appendix 1. As
mentioned earlier the sample size was not big, so these results do not necessarily apply to the whole population, to get more reliable results more patients should be used. This can be achieved by working together with different brain banks between different countries. All the researches will follow the same protocol, as shown in Appendix 2, and patients will be matched for potential confounding factors. This research only examined the brains of Narcolepsy type 1 patients, it cannot be said with certainty that the results also apply for Narcolepsy type 2 patients. Narcolepsy type 2 patients do not have an orexin reduction (Peyron et al., 2000 ; Thannickal et al., 2000), this only applies to Narcolepsy type 1, Narcolepsy type 2 patients may also not show an upregulation of HDC cells. To find out if they differ in HDC cell count another research can be conducted where the HDC cells of Narcolepsy type 2 patients will be examined. This can be performed by following the protocol that has been used in this HDC study, shown in Appendix 2.
This study has proven that Narcolepsy type 1 patients have an increased HDC cell count. This study has also shown a negative correlation between orexin and HDC in these patients, which was in agreement with the theory that orexin is destroyed by an inflammatory reaction. To answer the research question, Pitolisant reduces the EDS because it cancels out the effect of the increased HDC cell count in Narcolepsy type 2 patients. This leads to an increase in histamine and therefore a reduction in EDS.
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Appendix I. Protocol for H3R immunocytochemical staining
Deparaffinization and rehydrationXylene 2x10 min Ethanol 100% 2x5 min Ethanol 96% 1x5 min Ethanol 90% 1x5 min Ethanol 80% 1x5 min Ethanol 70% 1x5 min Ethanol 60% 1x5 min Ethanol 50% 1x5 min - Rinse the slides in aqua dest
- Wash the slides briefly (~2-3 min) in for antigen retrieval buffer pH7.6 - Antigen retrieval: Preheat sections at full power
- Microwave the slides in TBS (pH 7.6) for 2 times 5 mins
- Let the slides cool down at room temperature for 30 min in movement - Wash the slides in TBS 2x10 min
- Incubate the slides in rabbit IgG, anti-H3R 1:200 diluted in Sumi-milk 5% at RT for 6o min, then followed by overnight incubation at 40C in moist chamber
- Rinse the slides 3x10 min in TBS
- Incubate the slides in biotynilated anti-rabbit IgG 1:400 in Sumi at RT for 60 min (Prepare ABC complex (Vectastain, ABC elite kit) 1:800 in Sumi, 30 min before use) - Rinse the slides 3x10 min in TBS
- Incubate the slides in ABC-complex 1:800 in Sumi at RT for 60 min - Rinse the slides 3x10 min in TBS
- Incubate the slides in substrate solution for app. 10 min - Use Ni-solution
- Stop the reaction in TBS
- Dehydrate the slides in graded ethanols, 3-5 min each, clear in Xylene 2x10 min, coverslip with Entellan and dry the slides overnight
Stock solutions:
TBS 0.05M Tris, 0.15M NaCl, pH 7.6 (use HCl) TBS-0.5% milk TBS, 0.5% Elk milkpowder (w/v), pH 7.6
SUMI 0.05M Tris, 0.15M NaCl, 0.25% (w/v)gelatin, 0.5% Triton X-100 (v/v), pH 7.6 (use Hcl)
SUMI-0.5% milk SUMI, 0.5% Elk milkpowder (w/v), pH 7.6
Appendix II. Protocol for HDC immunocytochemical staining
- Slides are deparaffinized in xylene and rehydrated through a graded alcohol series. - Rinse the slides in aqua dest.- Microwave-treated with 0.01 M sodium citrate (pH=6.0; 2 x 5 minutes) - Let the slides cool down at room temperature for 30 min in movement - Wash the slides in TBS 2x10 min
- A one hour pre-incubation step in 5% TBS-milk (0.05 M Tris, 0.15 M NaCl, pH=7.6) at RT - Wash the slides in TBS 2*10 min
- TMN sections were incubated in rabbit-IgG, anti-HDC (EUD 2601, Acris) at a 1:4000 concentration in supermix-5% milk (0.25% gelatin, 0.5% Triton in TBS) at RT for an hour followed by overnight
incubation at 4°C in the moist chamber - Rinse the slides 3x10 min in TBS
- Incubate the slides in biotynilated anti-rabbit IgG 1:400 in Sumi at RT for 60 min (Prepare ABC complex (Vectastain, ABC elite kit) 1:800 in Sumi, 30 min before use) - Rinse the slides 3x10 min in TBS
- Incubate the slides in ABC-complex 1:800 in Sumi at RT for 60 min - Rinse the slides 2x 10 min in TBS
- Incubate the slides in substrate solution for app. 7 min - Use Ni-solution
- Stop the reaction in TBS
- Dehydrate the slides in graded ethanols, 3-5 min each, clear in Xylene 2x10 min, coverslip with Entellan and dry the slides overnight
Stock solutions:
TBS 0.05M Tris, 0.15M NaCl, pH 7.6 (use HCl) TBS-5% milk TBS, 0.5% Elk milkpowder (w/v), pH 7.6
SUMI 0.05M Tris, 0.15M NaCl, 0.25% (w/v)gelatin, 0.5% Triton X-100 (v/v), pH 7.6 (use Hcl)
SUMI-5% milk SUMI, 0.5% Elk milkpowder (w/v), pH 7.6
