Background The biological basis of pain in chronic pancreatitis is poorly understood. Mast cells have been implicated in the pathogenesis of pain in other conditions. We hypothesized that mast cells play a role in the pain of chronic pancreatitis. We examined the association of pain with mast cells in autopsy specimens of patients with painful chronic pancreatitis. We explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS) -induced chronic pancreatitis in both wild type (WT) and mast cell deficient mice (MCDM).
Background
The biological basis of pain in chronic pancreatitis is poorly understood. Mast cells have been implicated in the pathogenesis of pain in other conditions. We hypothesized that mast cells play a role in the pain of chronic pancreatitis.We examined the association of pain with mast cells in autopsy specimens of patients with painful chronic pancreatitis. We explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS) -induced chronic pancreatitis in both wild type (WT) and mast cell deficient mice (MCDM).Methods Archival tissues with histological diagnoses of chronic pancreatitis were identified and clinical records reviewed for presence or absence of reported pain in humans. Mast cells were counted. The presence of pain was assessed using von Frey Filaments (VFF) to measure abdominal withdrawal responses in both WT and MCDM mice with and without chronic pancreatitis.
Methods
Archival tissues with histological diagnoses of chronic pancreatitis were identified and clinical records reviewed for presence or absence of reported pain in humans. Mast cells were counted.The presence of pain was assessed using von Frey Filaments (VFF) to measure abdominal withdrawal responses in both WT and MCDM mice with and without chronic pancreatitis.Results Humans with painful chronic pancreatitis demonstrated a 3.5-fold increase in pancreatic mast cells as compared with those with painless chronic pancreatitis. WT mice with chronic pancreatitis were significantly more sensitive as assessed by VFF pain testing of the abdomen when compared with MCDM.
Results
Humans with painful chronic pancreatitis demonstrated a 3.5-fold increase in pancreatic mast cells as compared with those with painless chronic pancreatitis.WT mice with chronic pancreatitis were significantly more sensitive as assessed by VFF pain testing of the abdomen when compared with MCDM.Conclusion Humans with painful chronic pancreatitis have an increased number of pancreatic mast cells as compared with those with painless chronic pancreatitis. MCDM are less sensitive to mechanical stimulation of the abdomen after induction of chronic pancreatitis as compared with WT. Mast cells may play an important role in the pathogenesis of pain in chronic pancreatitis.
Conclusion
Humans with painful chronic pancreatitis have an increased number of pancreatic mast cells as compared with those with painless chronic pancreatitis. MCDM are less sensitive to mechanical stimulation of the abdomen after induction of chronic pancreatitis as compared with WT. Mast cells may play an important role in the pathogenesis of pain in chronic pancreatitis.

Background Although pain is the presenting symptom of most patients with chronic pancreatitis, its neurobiological basis remains poorly understood [ 1 ]. In the past, investigators have focused on the role of anatomical abnormalities such as a strictured pancreatic duct or narrowed intraparenchymal ducts. However, mechanical decompression techniques such as endoscopic stent placement or even surgical pancreatojejunostomy frequently do not provide a permanent solution to the pain [ 1 ]. More recently, investigators have begun focusing on the role of neurotransmitters and neurotrophins such as substance P and nerve growth factor with known or suspected roles in nociceptive signaling and/or sensitization and have reported an increased expression of several of them in the pancreas of patients with painful chronic pancreatitis [ 2 ]. Mast cells are also increased in both acute and chronic pancreatitis [ 3 , 4 ] but their role in the generation of pain in pancreatitis has not been investigated. We hypothesized that mast cells are involved in the pathogenesis of pain in chronic pancreatitis. This hypothesis is based on the following observations. First, mast cells have been associated with human conditions in which pain is a predominant symptom. Interstitial cystitis and irritable bowel syndrome are both conditions in which pain is out of proportion to the objective pathological findings [ 5 , 6 ]. In both conditions, an increase in the number of mast cells has been described in the bladder and the colon, respectively [ 5 , 6 ]. Further, mast cells are frequently found in close proximity to nerves in the intestinal mucosa and the bladder [ 7 - 9 ]. This has also been observed in the pancreas – the total number of mast cells was significantly higher in pancreatic tissue from patients with chronic pancreatitis than in the normal pancreatic controls [ 3 ]. One of the preferential locations of mast cells was around and within the perineurium of nerve fibers in tissue samples of patients with chronic pancreatitis, suggesting the potential for interactions between mast cells and the nervous system. Lastly, there is evidence for bi-directional functional communication between mast cells and nerves [ 10 - 12 ]. Mast cells can not only release mediators that increase excitability of neurons but in turn, neurotransmitters such as substance P can trigger mast cell degranulation [ 10 ]. Mast cells may therefore contribute to the pathogenesis of pain in pancreatitis through degranulation products that can sensitize pancreatic afferent neurons in an ongoing vicious circle of neuronally mediated mast cell degranulation. Our first aim was to analyze the presence and distribution of mast cells in autopsy specimens of chronic pancreatitis and study the correlation, if any, with historical documentation of pain. We then explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS)-induced chronic pancreatitis in both wild type and Kit W /Kit W-v  mice, a strain deficient in mast cells (MCDM).
Background
Although pain is the presenting symptom of most patients with chronic pancreatitis, its neurobiological basis remains poorly understood [ 1 ]. In the past, investigators have focused on the role of anatomical abnormalities such as a strictured pancreatic duct or narrowed intraparenchymal ducts. However, mechanical decompression techniques such as endoscopic stent placement or even surgical pancreatojejunostomy frequently do not provide a permanent solution to the pain [ 1 ]. More recently, investigators have begun focusing on the role of neurotransmitters and neurotrophins such as substance P and nerve growth factor with known or suspected roles in nociceptive signaling and/or sensitization and have reported an increased expression of several of them in the pancreas of patients with painful chronic pancreatitis [ 2 ]. Mast cells are also increased in both acute and chronic pancreatitis [ 3 , 4 ] but their role in the generation of pain in pancreatitis has not been investigated.We hypothesized that mast cells are involved in the pathogenesis of pain in chronic pancreatitis. This hypothesis is based on the following observations. First, mast cells have been associated with human conditions in which pain is a predominant symptom. Interstitial cystitis and irritable bowel syndrome are both conditions in which pain is out of proportion to the objective pathological findings [ 5 , 6 ]. In both conditions, an increase in the number of mast cells has been described in the bladder and the colon, respectively [ 5 , 6 ]. Further, mast cells are frequently found in close proximity to nerves in the intestinal mucosa and the bladder [ 7 - 9 ]. This has also been observed in the pancreas – the total number of mast cells was significantly higher in pancreatic tissue from patients with chronic pancreatitis than in the normal pancreatic controls [ 3 ]. One of the preferential locations of mast cells was around and within the perineurium of nerve fibers in tissue samples of patients with chronic pancreatitis, suggesting the potential for interactions between mast cells and the nervous system. Lastly, there is evidence for bi-directional functional communication between mast cells and nerves [ 10 - 12 ]. Mast cells can not only release mediators that increase excitability of neurons but in turn, neurotransmitters such as substance P can trigger mast cell degranulation [ 10 ]. Mast cells may therefore contribute to the pathogenesis of pain in pancreatitis through degranulation products that can sensitize pancreatic afferent neurons in an ongoing vicious circle of neuronally mediated mast cell degranulation.Our first aim was to analyze the presence and distribution of mast cells in autopsy specimens of chronic pancreatitis and study the correlation, if any, with historical documentation of pain. We then explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS)-induced chronic pancreatitis in both wild type and Kit W /Kit W-v  mice, a strain deficient in mast cells (MCDM).Methods Data collection autopsy study Autopsy records from the University of Texas Medical Branch from the years 1993 to 2000 were searched electronically for the term "pancreatitis." One-hundred sixty-six patients were identified of which 26 patients carried an autopsy diagnosis of chronic pancreatitis and 140 patients carried a diagnosis of acute pancreatitis. The medical charts from patients with an autopsy diagnosis of chronic pancreatitis were reviewed for documentation of a medical history of chronic pancreatitis. If no such documentation was present in the chart, patients were excluded from the study (12/26). Thus, 14/26 patients with both a documented history and an autopsy based diagnosis of chronic pancreatitis, were included in the study. Patients were categorized as painful chronic pancreatitis (8/26) when they fulfilled one of the following criteria: a documented history of chronic abdominal pain clinically attributed to chronic pancreatitis that required the use of narcotics, and/or frequent admissions for recurrent abdominal pain clinically attributed to chronic pancreatitis, and/or a surgical or endoscopic procedure for refractory abdominal pain clinically attributed to chronic pancreatitis. Patients were categorized as non-painful chronic pancreatitis (6/26) if patients did not fit any of the criteria listed under painful chronic pancreatitis. In addition, the following data were collected: demographic factors (age and race), cause of death, comorbidities, clinical history of pancreatitis, etiology of pancreatitis, diagnostic studies supporting a diagnosis of pancreatitis (amylase, lipase, calcifications on abdominal plain film, CT-scan, ultrasound or ERCP). Human pancreatic control tissue was obtained from 8 arbitrarily chosen patients of whom the autopsy records recorded acute myocardial infarction as the cause of death. Their medical records were reviewed to ensure that they did not have a clinical history of pancreatitis. Therefore there were three categories of patients: one with painful chronic pancreatitis, one with non-painful chronic pancreatitis and non-pancreatitis controls. A pathologist, blinded to the group assignment, verified all histological diagnoses and counted mast cells on a Giemsa stained tissue section (average of 10 high-power randomly chosen (40X) fields per specimen). The protocol was approved by the Institutional Review Board of the University of Texas Medical Branch. Mice strains All mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Male mice were used from the following strain: WBB6F1/j-Kit W /Kit W-v  (MCDM) and the respective littermate control mouse strain, Kit W-v  – +/+ (WT). The mice were 3 months of age at the onset of the experiment with body weights of 25–30 gram. Experimental protocols involving mice were approved by our Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines provided by the National Institutes of Health. Induction of chronic pancreatitis Mice were anesthetized with sodium Nembutal (50 mg/kg body weigh, i.p.) Following a midabdominal laparotomy, a canula was introduced into the common pancreato-biliary duct; the duct was ligated proximally and distally to ensure perfusion into the pancreas and prevent entry of the injected substance into the liver or duodenum. 0.1 ml of 1% TNBS in phosphate buffered saline (PBS)-10% ethanol, pH 8, was infused into the pancreas (modified after Puig-Divi [ 13 ]). The canula was removed and the abdomen closed. Control mice were treated in the exact same fashion but were perfused with saline instead (Figure  1 ). Mice were sacrificed 8 weeks after surgery. Von Frey Filament (VFF) measurements VFF hairs consist of a series of filaments of increasing diameter that produce increasing sensations of touch when applied to the skin. When the tip of a fiber of given length and diameter is pressed against the skin, the force of application increases until the fiber bends. After the fiber bends, continued advance creates more bend, but not more force of application. This principle makes it possible to apply a reproducible force to the skin surface. VFF testing is an established behavioral pain assay used to determine mechanical pain thresholds in somatic pain. More recently, VFF testing has been used as a surrogate marker for visceral pain [ 14 , 15 ]. Mice were placed in a cage with a mesh floor and habituated to the environment for 30–60 minutes. Measurements were taken from the abdomen and the plantar surface of both hindpaws over a period of three weeks prior to the surgery and for a total of three weeks after the surgery (Figure  1 ). VFF filaments of various caliber were applied to the mid-abdomen in ascending order 10 times, each for 1–2 seconds with a 10 second interval. A response was defined as: a) sharp retraction of the abdomen; b) immediate licking or scratching of site of application of hair; or c) jumping. The response frequency was defined as the total number of responses out of 10 applications (expressed as a percentage) to the skin per filament. An investigator blinded to the different treatment groups performed the behavioral testing. Pancreatic histology Fresh specimens of the mouse pancreas were fixed in 10% formaldehyde in PBS pH 7.4 containing 1 mM MgCl 2  at 4°C overnight. Sections from paraffin-embedded specimens were stained with hematoxyline and eosin and observed under a light microscope. Pathological changes were scored based on a scale described by Tito et al. by a pathologist blinded to the different treatment groups [ 16 ]. Data expression and statistical analysis Comparisons of the number of mast cells in autopsy specimen were analyzed using the Mann-Whitney U test. For each behavioral experiment (see figure  1 ), the average response frequency was calculated as the mean of the mean response frequencies for each mouse (across four measures). The "post-pre response frequency" was calculated by subtracting the pre-surgical average response frequency from the post-surgical average response frequency. To assess the independent effect of TNBS on VFF response (ie. to control for the effect of the surgery itself), the post-pre response frequency for TNBS infusion was compared with the post-pre response frequency for saline infusion. This comparison was performed using analysis of variance for a two-factor experiment with repeated measures on time at each level of force for each type of mice (WT and MCDM). The two factors were induction of pancreatitis or not (TNBS or saline, respectively) and time (pre-surgical or post-surgical). TNBS infusion was considered to have had an independent effect on the VFF response if the post-pre response frequency was greater for TNBS than for saline infusion. Fisher's least significant difference procedure was used for multiple comparisons of least squares means with Bonferroni adjustment for number of comparisons. All effects and interactions were assessed at the 0.05 level of significance. Data analysis was conducted using PROC MIXED with LSMEANS options in SAS ® , Release 8.2 [SAS Institute Inc., SAS/STAT ®  User's Guide, Version 8, Cary, NC: SAS Institute Inc., 1999].
Methods
Data collection autopsy study Autopsy records from the University of Texas Medical Branch from the years 1993 to 2000 were searched electronically for the term "pancreatitis." One-hundred sixty-six patients were identified of which 26 patients carried an autopsy diagnosis of chronic pancreatitis and 140 patients carried a diagnosis of acute pancreatitis. The medical charts from patients with an autopsy diagnosis of chronic pancreatitis were reviewed for documentation of a medical history of chronic pancreatitis. If no such documentation was present in the chart, patients were excluded from the study (12/26). Thus, 14/26 patients with both a documented history and an autopsy based diagnosis of chronic pancreatitis, were included in the study. Patients were categorized as painful chronic pancreatitis (8/26) when they fulfilled one of the following criteria: a documented history of chronic abdominal pain clinically attributed to chronic pancreatitis that required the use of narcotics, and/or frequent admissions for recurrent abdominal pain clinically attributed to chronic pancreatitis, and/or a surgical or endoscopic procedure for refractory abdominal pain clinically attributed to chronic pancreatitis. Patients were categorized as non-painful chronic pancreatitis (6/26) if patients did not fit any of the criteria listed under painful chronic pancreatitis. In addition, the following data were collected: demographic factors (age and race), cause of death, comorbidities, clinical history of pancreatitis, etiology of pancreatitis, diagnostic studies supporting a diagnosis of pancreatitis (amylase, lipase, calcifications on abdominal plain film, CT-scan, ultrasound or ERCP). Human pancreatic control tissue was obtained from 8 arbitrarily chosen patients of whom the autopsy records recorded acute myocardial infarction as the cause of death. Their medical records were reviewed to ensure that they did not have a clinical history of pancreatitis. Therefore there were three categories of patients: one with painful chronic pancreatitis, one with non-painful chronic pancreatitis and non-pancreatitis controls. A pathologist, blinded to the group assignment, verified all histological diagnoses and counted mast cells on a Giemsa stained tissue section (average of 10 high-power randomly chosen (40X) fields per specimen). The protocol was approved by the Institutional Review Board of the University of Texas Medical Branch.
Data collection autopsy study
Autopsy records from the University of Texas Medical Branch from the years 1993 to 2000 were searched electronically for the term "pancreatitis." One-hundred sixty-six patients were identified of which 26 patients carried an autopsy diagnosis of chronic pancreatitis and 140 patients carried a diagnosis of acute pancreatitis. The medical charts from patients with an autopsy diagnosis of chronic pancreatitis were reviewed for documentation of a medical history of chronic pancreatitis. If no such documentation was present in the chart, patients were excluded from the study (12/26). Thus, 14/26 patients with both a documented history and an autopsy based diagnosis of chronic pancreatitis, were included in the study. Patients were categorized as painful chronic pancreatitis (8/26) when they fulfilled one of the following criteria: a documented history of chronic abdominal pain clinically attributed to chronic pancreatitis that required the use of narcotics, and/or frequent admissions for recurrent abdominal pain clinically attributed to chronic pancreatitis, and/or a surgical or endoscopic procedure for refractory abdominal pain clinically attributed to chronic pancreatitis. Patients were categorized as non-painful chronic pancreatitis (6/26) if patients did not fit any of the criteria listed under painful chronic pancreatitis. In addition, the following data were collected: demographic factors (age and race), cause of death, comorbidities, clinical history of pancreatitis, etiology of pancreatitis, diagnostic studies supporting a diagnosis of pancreatitis (amylase, lipase, calcifications on abdominal plain film, CT-scan, ultrasound or ERCP). Human pancreatic control tissue was obtained from 8 arbitrarily chosen patients of whom the autopsy records recorded acute myocardial infarction as the cause of death. Their medical records were reviewed to ensure that they did not have a clinical history of pancreatitis. Therefore there were three categories of patients: one with painful chronic pancreatitis, one with non-painful chronic pancreatitis and non-pancreatitis controls. A pathologist, blinded to the group assignment, verified all histological diagnoses and counted mast cells on a Giemsa stained tissue section (average of 10 high-power randomly chosen (40X) fields per specimen). The protocol was approved by the Institutional Review Board of the University of Texas Medical Branch.Mice strains All mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Male mice were used from the following strain: WBB6F1/j-Kit W /Kit W-v  (MCDM) and the respective littermate control mouse strain, Kit W-v  – +/+ (WT). The mice were 3 months of age at the onset of the experiment with body weights of 25–30 gram. Experimental protocols involving mice were approved by our Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines provided by the National Institutes of Health.
Mice strains
All mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Male mice were used from the following strain: WBB6F1/j-Kit W /Kit W-v  (MCDM) and the respective littermate control mouse strain, Kit W-v  – +/+ (WT). The mice were 3 months of age at the onset of the experiment with body weights of 25–30 gram.Experimental protocols involving mice were approved by our Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines provided by the National Institutes of Health.Induction of chronic pancreatitis Mice were anesthetized with sodium Nembutal (50 mg/kg body weigh, i.p.) Following a midabdominal laparotomy, a canula was introduced into the common pancreato-biliary duct; the duct was ligated proximally and distally to ensure perfusion into the pancreas and prevent entry of the injected substance into the liver or duodenum. 0.1 ml of 1% TNBS in phosphate buffered saline (PBS)-10% ethanol, pH 8, was infused into the pancreas (modified after Puig-Divi [ 13 ]). The canula was removed and the abdomen closed. Control mice were treated in the exact same fashion but were perfused with saline instead (Figure  1 ). Mice were sacrificed 8 weeks after surgery.
Induction of chronic pancreatitis
Mice were anesthetized with sodium Nembutal (50 mg/kg body weigh, i.p.) Following a midabdominal laparotomy, a canula was introduced into the common pancreato-biliary duct; the duct was ligated proximally and distally to ensure perfusion into the pancreas and prevent entry of the injected substance into the liver or duodenum. 0.1 ml of 1% TNBS in phosphate buffered saline (PBS)-10% ethanol, pH 8, was infused into the pancreas (modified after Puig-Divi [ 13 ]). The canula was removed and the abdomen closed. Control mice were treated in the exact same fashion but were perfused with saline instead (Figure  1 ). Mice were sacrificed 8 weeks after surgery.Von Frey Filament (VFF) measurements VFF hairs consist of a series of filaments of increasing diameter that produce increasing sensations of touch when applied to the skin. When the tip of a fiber of given length and diameter is pressed against the skin, the force of application increases until the fiber bends. After the fiber bends, continued advance creates more bend, but not more force of application. This principle makes it possible to apply a reproducible force to the skin surface. VFF testing is an established behavioral pain assay used to determine mechanical pain thresholds in somatic pain. More recently, VFF testing has been used as a surrogate marker for visceral pain [ 14 , 15 ]. Mice were placed in a cage with a mesh floor and habituated to the environment for 30–60 minutes. Measurements were taken from the abdomen and the plantar surface of both hindpaws over a period of three weeks prior to the surgery and for a total of three weeks after the surgery (Figure  1 ). VFF filaments of various caliber were applied to the mid-abdomen in ascending order 10 times, each for 1–2 seconds with a 10 second interval. A response was defined as: a) sharp retraction of the abdomen; b) immediate licking or scratching of site of application of hair; or c) jumping. The response frequency was defined as the total number of responses out of 10 applications (expressed as a percentage) to the skin per filament. An investigator blinded to the different treatment groups performed the behavioral testing.
Von Frey Filament (VFF) measurements
VFF hairs consist of a series of filaments of increasing diameter that produce increasing sensations of touch when applied to the skin. When the tip of a fiber of given length and diameter is pressed against the skin, the force of application increases until the fiber bends. After the fiber bends, continued advance creates more bend, but not more force of application. This principle makes it possible to apply a reproducible force to the skin surface. VFF testing is an established behavioral pain assay used to determine mechanical pain thresholds in somatic pain. More recently, VFF testing has been used as a surrogate marker for visceral pain [ 14 , 15 ].Mice were placed in a cage with a mesh floor and habituated to the environment for 30–60 minutes. Measurements were taken from the abdomen and the plantar surface of both hindpaws over a period of three weeks prior to the surgery and for a total of three weeks after the surgery (Figure  1 ). VFF filaments of various caliber were applied to the mid-abdomen in ascending order 10 times, each for 1–2 seconds with a 10 second interval. A response was defined as: a) sharp retraction of the abdomen; b) immediate licking or scratching of site of application of hair; or c) jumping. The response frequency was defined as the total number of responses out of 10 applications (expressed as a percentage) to the skin per filament. An investigator blinded to the different treatment groups performed the behavioral testing.Pancreatic histology Fresh specimens of the mouse pancreas were fixed in 10% formaldehyde in PBS pH 7.4 containing 1 mM MgCl 2  at 4°C overnight. Sections from paraffin-embedded specimens were stained with hematoxyline and eosin and observed under a light microscope. Pathological changes were scored based on a scale described by Tito et al. by a pathologist blinded to the different treatment groups [ 16 ].
Pancreatic histology
Fresh specimens of the mouse pancreas were fixed in 10% formaldehyde in PBS pH 7.4 containing 1 mM MgCl 2  at 4°C overnight. Sections from paraffin-embedded specimens were stained with hematoxyline and eosin and observed under a light microscope. Pathological changes were scored based on a scale described by Tito et al. by a pathologist blinded to the different treatment groups [ 16 ].Data expression and statistical analysis Comparisons of the number of mast cells in autopsy specimen were analyzed using the Mann-Whitney U test. For each behavioral experiment (see figure  1 ), the average response frequency was calculated as the mean of the mean response frequencies for each mouse (across four measures). The "post-pre response frequency" was calculated by subtracting the pre-surgical average response frequency from the post-surgical average response frequency. To assess the independent effect of TNBS on VFF response (ie. to control for the effect of the surgery itself), the post-pre response frequency for TNBS infusion was compared with the post-pre response frequency for saline infusion. This comparison was performed using analysis of variance for a two-factor experiment with repeated measures on time at each level of force for each type of mice (WT and MCDM). The two factors were induction of pancreatitis or not (TNBS or saline, respectively) and time (pre-surgical or post-surgical). TNBS infusion was considered to have had an independent effect on the VFF response if the post-pre response frequency was greater for TNBS than for saline infusion. Fisher's least significant difference procedure was used for multiple comparisons of least squares means with Bonferroni adjustment for number of comparisons. All effects and interactions were assessed at the 0.05 level of significance. Data analysis was conducted using PROC MIXED with LSMEANS options in SAS ® , Release 8.2 [SAS Institute Inc., SAS/STAT ®  User's Guide, Version 8, Cary, NC: SAS Institute Inc., 1999].
Data expression and statistical analysis
Comparisons of the number of mast cells in autopsy specimen were analyzed using the Mann-Whitney U test.For each behavioral experiment (see figure  1 ), the average response frequency was calculated as the mean of the mean response frequencies for each mouse (across four measures). The "post-pre response frequency" was calculated by subtracting the pre-surgical average response frequency from the post-surgical average response frequency. To assess the independent effect of TNBS on VFF response (ie. to control for the effect of the surgery itself), the post-pre response frequency for TNBS infusion was compared with the post-pre response frequency for saline infusion. This comparison was performed using analysis of variance for a two-factor experiment with repeated measures on time at each level of force for each type of mice (WT and MCDM). The two factors were induction of pancreatitis or not (TNBS or saline, respectively) and time (pre-surgical or post-surgical). TNBS infusion was considered to have had an independent effect on the VFF response if the post-pre response frequency was greater for TNBS than for saline infusion.Fisher's least significant difference procedure was used for multiple comparisons of least squares means with Bonferroni adjustment for number of comparisons. All effects and interactions were assessed at the 0.05 level of significance. Data analysis was conducted using PROC MIXED with LSMEANS options in SAS ® , Release 8.2 [SAS Institute Inc., SAS/STAT ®  User's Guide, Version 8, Cary, NC: SAS Institute Inc., 1999].Results Autopsy data Patient demographics are summarized in Table  1 . Alcohol abuse was the most common cause for pancreatitis in both groups. Analysis of our results, using the Mann-Whitney U test, revealed significantly more mast cells in patients with a history of painful chronic pancreatitis (n = 8) when compared to patients with either non-painful chronic pancreatitis (n = 6) (33.8 vs 9.4 average mast cell count/10 high power fields; p < 0.01) or controls (n = 8), (33.8 vs 6.1 average mast cell count/10 high power fields; p < 0.01) (Figure  2 ). The increased number of mast cells in patients with painful pancreatitis was noted predominantly in interstitial areas and, to a lesser degree, in the periacinar space. Withdrawal responses to mechanical stimulation of the abdomen and paw after induction of chronic pancreatitis Figure  3  shows the post-pre surgical response frequency for both WT and MCDM. TNBS had a significant independent effect on abdominal VFF response in WT mice at the force levels 4 and 8 mN (p = 0.007 and 0.037, respectively) (Figure  3A ). There was a trend towards a significant effect at the force level of 16 mN (p = 0.066). In contrast, for MCDM, TNBS had no significant effect on abdominal VFF response at any force level (Figure  3B ). There was no significant TNBS effect on VFF response in the left hindpaw for either WT mice or MCDM (Figure  4 ). Data not shown for the right hindpaw. Histological analysis of mice pancreas following TNBS injection Pancreatic histology confirmed the presence of chronic pancreatitis in both WT and MCDM with marked fibrosis, inflammatory infiltrates and ductular proliferation mimicking changes seen in human chronic pancreatitis (Figure  5A ). The pancreas of saline treated controls was normal. There was no significant difference in the overall inflammatory scores between the WT and MCDM (Figure  5B ). An increased number of mast cells were counted in WT mice with chronic pancreatitis compared to saline treated controls (5.6 vs 1.5; p = 0.05) (Figure  6 ). As to be expected, no mast cells were present in pancreas of MCDM.
Results
Autopsy data Patient demographics are summarized in Table  1 . Alcohol abuse was the most common cause for pancreatitis in both groups. Analysis of our results, using the Mann-Whitney U test, revealed significantly more mast cells in patients with a history of painful chronic pancreatitis (n = 8) when compared to patients with either non-painful chronic pancreatitis (n = 6) (33.8 vs 9.4 average mast cell count/10 high power fields; p < 0.01) or controls (n = 8), (33.8 vs 6.1 average mast cell count/10 high power fields; p < 0.01) (Figure  2 ). The increased number of mast cells in patients with painful pancreatitis was noted predominantly in interstitial areas and, to a lesser degree, in the periacinar space.
Autopsy data
Patient demographics are summarized in Table  1 . Alcohol abuse was the most common cause for pancreatitis in both groups. Analysis of our results, using the Mann-Whitney U test, revealed significantly more mast cells in patients with a history of painful chronic pancreatitis (n = 8) when compared to patients with either non-painful chronic pancreatitis (n = 6) (33.8 vs 9.4 average mast cell count/10 high power fields; p < 0.01) or controls (n = 8), (33.8 vs 6.1 average mast cell count/10 high power fields; p < 0.01) (Figure  2 ). The increased number of mast cells in patients with painful pancreatitis was noted predominantly in interstitial areas and, to a lesser degree, in the periacinar space.Withdrawal responses to mechanical stimulation of the abdomen and paw after induction of chronic pancreatitis Figure  3  shows the post-pre surgical response frequency for both WT and MCDM. TNBS had a significant independent effect on abdominal VFF response in WT mice at the force levels 4 and 8 mN (p = 0.007 and 0.037, respectively) (Figure  3A ). There was a trend towards a significant effect at the force level of 16 mN (p = 0.066). In contrast, for MCDM, TNBS had no significant effect on abdominal VFF response at any force level (Figure  3B ). There was no significant TNBS effect on VFF response in the left hindpaw for either WT mice or MCDM (Figure  4 ). Data not shown for the right hindpaw.
Withdrawal responses to mechanical stimulation of the abdomen and paw after induction of chronic pancreatitis
Figure  3  shows the post-pre surgical response frequency for both WT and MCDM. TNBS had a significant independent effect on abdominal VFF response in WT mice at the force levels 4 and 8 mN (p = 0.007 and 0.037, respectively) (Figure  3A ). There was a trend towards a significant effect at the force level of 16 mN (p = 0.066). In contrast, for MCDM, TNBS had no significant effect on abdominal VFF response at any force level (Figure  3B ). There was no significant TNBS effect on VFF response in the left hindpaw for either WT mice or MCDM (Figure  4 ). Data not shown for the right hindpaw.Histological analysis of mice pancreas following TNBS injection Pancreatic histology confirmed the presence of chronic pancreatitis in both WT and MCDM with marked fibrosis, inflammatory infiltrates and ductular proliferation mimicking changes seen in human chronic pancreatitis (Figure  5A ). The pancreas of saline treated controls was normal. There was no significant difference in the overall inflammatory scores between the WT and MCDM (Figure  5B ). An increased number of mast cells were counted in WT mice with chronic pancreatitis compared to saline treated controls (5.6 vs 1.5; p = 0.05) (Figure  6 ). As to be expected, no mast cells were present in pancreas of MCDM.
Histological analysis of mice pancreas following TNBS injection
Pancreatic histology confirmed the presence of chronic pancreatitis in both WT and MCDM with marked fibrosis, inflammatory infiltrates and ductular proliferation mimicking changes seen in human chronic pancreatitis (Figure  5A ). The pancreas of saline treated controls was normal. There was no significant difference in the overall inflammatory scores between the WT and MCDM (Figure  5B ). An increased number of mast cells were counted in WT mice with chronic pancreatitis compared to saline treated controls (5.6 vs 1.5; p = 0.05) (Figure  6 ). As to be expected, no mast cells were present in pancreas of MCDM.Discussion Chronic pancreatitis has been defined as a continuing inflammatory disease of the pancreas characterized by irreversible morphologic changes that typically cause pain and/or permanent loss of function [ 17 ]. The pathogenesis of pain in this condition remains to be satisfactorily established. We examined the association, if any, of pain with mast cells as quantified in autopsy specimens of patients with a history of painful and non-painful chronic pancreatitis and normal controls. Significantly more mast cells were present in pancreatic tissue from patients with a history of painful chronic pancreatitis, indicating an association with this condition and a potential role for these cells in the pathogenesis of pain in painful chronic pancreatitis. There are clearly limitations to a retrospective, autopsy-based study such as the one we report here. For instance, we do not know whether pain was present at the time of death and there was incomplete information on the different patterns of pain. Also, our findings pertain mainly to patients with a history of alcoholic pancreatitis. Nevertheless, our findings do suggest an association of painful chronic pancreatitis with an increased number of mast cells. This observation provided the rationale for further experimental testing, which we performed in mice. We first developed a model of chronic pancreatitis in mice following a modified protocol first described by Puig- Divi  et al . [ 13 ]. Histological changes consisted of periductal and lobular fibrosis, duct stenosis, chronic inflammatory cell infiltrates, and gland atrophy, mimicking features of chronic pancreatitis in humans. Significantly more mast cells were present in WT mice with chronic pancreatitis, adding to the validity of this model for use in studies on the role of mast cells in pancreatitis. Both WT and MCDM developed histological changes consistent with chronic pancreatitis, indicating that the elimination of mast cells did not modulate the animals' ability to mount an inflammatory response. Therefore, any changes observed in pain behavior are unlikely to stem from differences in underlying inflammation. Next we determined whether this mouse model could be used to study behavioral differences associated with chronic pancreatitis. The assessment of spontaneous pain in a visceral organ presents significant difficulties. We have used a behavioral method to assess this, which relies on the association of visceral pain with sensitization of somatic regions of the body that share segmental innervation at the level of the spinal cord (referred pain). This somatic sensitization can be quantified using VFF to stimulate the somatotopically appropriate abdominal region and measuring the abdominal withdrawal response. Thus, VFF testing of the anterior abdominal wall can be used as a surrogate marker for visceral pain. Although this is the first time that this technique has been used for the measurement of referred visceral hyperalgesia in a mouse model of chronic pancreatitis, this method has previously been described and validated to assess the severity of referred visceral pain for models of colonic hypersensitivity [ 14 ] as well as rat models of acute necrotizing pancreatitis [ 15 ] and chronic pancreatitis [ 18 ]. The abdominal VFF response was compared to the hind paw response to assess the specificity of the interventions to the pancreas. TNBS treated mice, but not the saline control, developed increased abdominal wall withdrawal responses to VFF testing when compared to baseline, suggesting the development of force-dependent referred hyperalgesia of the abdominal wall in WT mice. There was no evidence of referred hyperalgesia in the hindpaws, suggesting that the measured effect on abdominal withdrawal is specific for an intra-abdominal origin of the pain. Vera-Portocarrero et al. previously described similar findings, increased withdrawal frequency after VFF stimulation to the abdominal area, in a rat model of chronic pancreatitis [ 18 ]. These behavioral changes were abrogated by morphine. Rats that demonstrated behavioral changes also expressed increased substance P expression in the nociceptive layers of the spinal cord, suggestive of central nociceptive changes. Mast cells produce a variety of degranulation products in the setting of inflammation that may activate and/or sensitize primary nociceptive neurons. The neurotrophin growth factor (NGF) is one such product [ 19 - 22 ]. NGF is released in the setting of inflammation and can not only function as a chemoattractant for other mast cells, but it can also trigger mast cell degranulation [ 23 ]. We are speculating that NGF production in the inflamed pancreas is responsible for plastic changes in the sensory neurons by activating proalgesic receptors and channels such as the NGF receptor tyrosine kinase A (TrkA) and Transient Receptor Potential Family V receptor 1 (TRPV1; previously known as VR1) thereby contributing to the generation of pain [ 24 - 26 ]. Similarly, other mast cell degranulation products such as tryptase and histamine are capable of modulating neuronal function [ 27 - 32 ]. Tryptase may directly activate the proteinase-activated receptor-2 (PAR-2), a G-protein coupled receptor expressed by pancreatic nerves, important in the pathogenesis of pain in pancreatitis [ 33 , 34 ]. Although the role for mast cells in the mediation of visceral nociceptive signaling needs to be explored further, we speculate that mast cell products released in pancreatitis, contribute to the development of pain by direct effects on nociceptors located on pancreatic afferent neurons (Figure  7 ). However, before concluding a definite role for mast cells from our experimental data, it should be noted that MCDM carry a spontaneous mutation for tyrosine kinase receptor c-kit which not only produces a deficiency of mast cells but may have an independent effect on the function of sensory neurons, which are known to express it [ 35 ]. Therefore, it remains to be determined whether the detected differences in nociceptive responses is due to the absence of mast cells per se or a yet unknown change in the responsiveness of sensory neurons due to a congenital lack of the c-kit receptor. Reconstitution of mast cells into the MCDM mice should restore their nociceptive responses close to the wild type phenotype.
Discussion
Chronic pancreatitis has been defined as a continuing inflammatory disease of the pancreas characterized by irreversible morphologic changes that typically cause pain and/or permanent loss of function [ 17 ]. The pathogenesis of pain in this condition remains to be satisfactorily established. We examined the association, if any, of pain with mast cells as quantified in autopsy specimens of patients with a history of painful and non-painful chronic pancreatitis and normal controls. Significantly more mast cells were present in pancreatic tissue from patients with a history of painful chronic pancreatitis, indicating an association with this condition and a potential role for these cells in the pathogenesis of pain in painful chronic pancreatitis.There are clearly limitations to a retrospective, autopsy-based study such as the one we report here. For instance, we do not know whether pain was present at the time of death and there was incomplete information on the different patterns of pain. Also, our findings pertain mainly to patients with a history of alcoholic pancreatitis. Nevertheless, our findings do suggest an association of painful chronic pancreatitis with an increased number of mast cells. This observation provided the rationale for further experimental testing, which we performed in mice. We first developed a model of chronic pancreatitis in mice following a modified protocol first described by Puig- Divi  et al . [ 13 ]. Histological changes consisted of periductal and lobular fibrosis, duct stenosis, chronic inflammatory cell infiltrates, and gland atrophy, mimicking features of chronic pancreatitis in humans. Significantly more mast cells were present in WT mice with chronic pancreatitis, adding to the validity of this model for use in studies on the role of mast cells in pancreatitis. Both WT and MCDM developed histological changes consistent with chronic pancreatitis, indicating that the elimination of mast cells did not modulate the animals' ability to mount an inflammatory response. Therefore, any changes observed in pain behavior are unlikely to stem from differences in underlying inflammation.et alNext we determined whether this mouse model could be used to study behavioral differences associated with chronic pancreatitis. The assessment of spontaneous pain in a visceral organ presents significant difficulties. We have used a behavioral method to assess this, which relies on the association of visceral pain with sensitization of somatic regions of the body that share segmental innervation at the level of the spinal cord (referred pain). This somatic sensitization can be quantified using VFF to stimulate the somatotopically appropriate abdominal region and measuring the abdominal withdrawal response. Thus, VFF testing of the anterior abdominal wall can be used as a surrogate marker for visceral pain. Although this is the first time that this technique has been used for the measurement of referred visceral hyperalgesia in a mouse model of chronic pancreatitis, this method has previously been described and validated to assess the severity of referred visceral pain for models of colonic hypersensitivity [ 14 ] as well as rat models of acute necrotizing pancreatitis [ 15 ] and chronic pancreatitis [ 18 ]. The abdominal VFF response was compared to the hind paw response to assess the specificity of the interventions to the pancreas. TNBS treated mice, but not the saline control, developed increased abdominal wall withdrawal responses to VFF testing when compared to baseline, suggesting the development of force-dependent referred hyperalgesia of the abdominal wall in WT mice. There was no evidence of referred hyperalgesia in the hindpaws, suggesting that the measured effect on abdominal withdrawal is specific for an intra-abdominal origin of the pain. Vera-Portocarrero et al. previously described similar findings, increased withdrawal frequency after VFF stimulation to the abdominal area, in a rat model of chronic pancreatitis [ 18 ]. These behavioral changes were abrogated by morphine. Rats that demonstrated behavioral changes also expressed increased substance P expression in the nociceptive layers of the spinal cord, suggestive of central nociceptive changes.Mast cells produce a variety of degranulation products in the setting of inflammation that may activate and/or sensitize primary nociceptive neurons. The neurotrophin growth factor (NGF) is one such product [ 19 - 22 ]. NGF is released in the setting of inflammation and can not only function as a chemoattractant for other mast cells, but it can also trigger mast cell degranulation [ 23 ]. We are speculating that NGF production in the inflamed pancreas is responsible for plastic changes in the sensory neurons by activating proalgesic receptors and channels such as the NGF receptor tyrosine kinase A (TrkA) and Transient Receptor Potential Family V receptor 1 (TRPV1; previously known as VR1) thereby contributing to the generation of pain [ 24 - 26 ]. Similarly, other mast cell degranulation products such as tryptase and histamine are capable of modulating neuronal function [ 27 - 32 ]. Tryptase may directly activate the proteinase-activated receptor-2 (PAR-2), a G-protein coupled receptor expressed by pancreatic nerves, important in the pathogenesis of pain in pancreatitis [ 33 , 34 ]. Although the role for mast cells in the mediation of visceral nociceptive signaling needs to be explored further, we speculate that mast cell products released in pancreatitis, contribute to the development of pain by direct effects on nociceptors located on pancreatic afferent neurons (Figure  7 ).However, before concluding a definite role for mast cells from our experimental data, it should be noted that MCDM carry a spontaneous mutation for tyrosine kinase receptor c-kit which not only produces a deficiency of mast cells but may have an independent effect on the function of sensory neurons, which are known to express it [ 35 ]. Therefore, it remains to be determined whether the detected differences in nociceptive responses is due to the absence of mast cells per se or a yet unknown change in the responsiveness of sensory neurons due to a congenital lack of the c-kit receptor. Reconstitution of mast cells into the MCDM mice should restore their nociceptive responses close to the wild type phenotype.Conclusion Our data should increase awareness of the importance of mast cells in the pathogenesis of painful inflammatory conditions such as chronic pancreatitis and encourage experimental studies for further testing of this hypothesis.
Conclusion
Our data should increase awareness of the importance of mast cells in the pathogenesis of painful inflammatory conditions such as chronic pancreatitis and encourage experimental studies for further testing of this hypothesis.Abbreviations TNBS – trinitrobenzene sulfonic acid, WT – wild type, MCDM – mast cell deficient mice, VFF – von Frey Filaments, IACUC – Institutional Animal Care and Use Committee, PBS – phosphate buffered saline, NGF – neurotrophin growth factor, TrkA – tyrosine kinase A, TRPV1 – Transient Receptor Potential Family V receptor 1 (previously known as VR1), PAR-2 – proteinase-activated receptor-2
Abbreviations
TNBS – trinitrobenzene sulfonic acid, WT – wild type, MCDM – mast cell deficient mice, VFF – von Frey Filaments, IACUC – Institutional Animal Care and Use Committee, PBS – phosphate buffered saline, NGF – neurotrophin growth factor, TrkA – tyrosine kinase A, TRPV1 – Transient Receptor Potential Family V receptor 1 (previously known as VR1), PAR-2 – proteinase-activated receptor-2Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions WAH conceived of the study, participated in its design and coordination and wrote the manuscript. KG completed the behavioral studies. SYX completed the histological analyses and mast cell counts. JHW provided technical assistance with the surgical procedures and participated in the data analyses and review of the manuscript. WDW provided support for the behavioral studies as well as their analyses. PJP provided financial support and participated in the review of the manuscript.
Authors' contributions
WAH conceived of the study, participated in its design and coordination and wrote the manuscript. KG completed the behavioral studies. SYX completed the histological analyses and mast cell counts. JHW provided technical assistance with the surgical procedures and participated in the data analyses and review of the manuscript. WDW provided support for the behavioral studies as well as their analyses. PJP provided financial support and participated in the review of the manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


In the 2002 thriller  Memento , the protagonist, Lenny, is plagued by a crippling neurological disorder that renders him incapable of storing memories for longer than fifteen minutes. He wants nothing more than to avenge the murder of his wife, but as another character in the film tells him, “Even if you get revenge, you're not going to remember it. You're not even going to know it happened.” Lenny has anterograde amnesia, a condition typically caused by stroke or other illness. (In the film, it's caused by a blow to the head.) Most forms of amnesia—including the more common retrograde amnesia, which involves the loss of long-term memory—are caused by some type of brain injury— particularly to the hippocampus.MementoRetrograde amnesia can arise from brain damage that interferes with memory storage, retrieval, or consolidation. And it is by studying retrograde amnesia that scientists have developed theories explaining where the brain stores memory traces and how it consolidates them into long-term memories. What ultimately causes amnesia—a failure to store memories or a failure to retrieve them—is not clear.A major challenge in resolving this question experimentally is being able to determine whether an animal has truly recovered a lost memory or has simply re-learned the task at hand. By refining a classic behavioral neuroscience experiment to test spatial learning and memory, Livia de Hoz, Stephen Martin, and Richard Morris have developed a novel protocol that distinguishes retrieval from new learning. The experiment, developed by Morris over fifteen years ago, takes advantage of the fact that rats are good swimmers but prefer life out of water. Rats are placed in a pool and trained to remember the location of an escape platform, hidden just under the surface. (The rats can't see it because the water is cloudy.)Here, de Hoz et al. trained rats to a particular platform location—their escape route—then gave them hippocampal lesions. One group received “sham” lesions, another partial lesions, and a third complete lesions. Partial lesions, the authors explain, should damage only a subset of the stored memory traces and thus weaken the rats' memory rather than completely disrupting it.The rats' postoperative memory was tested by first placing them in the pool with the platform hidden: as expected, the lesioned rats couldn't remember where it was. After a minute, the platform was raised above the water, to remind them that it existed and that they could escape by climbing onto it. The platform was revealed in the original location for half the animals and in a novel location—which could be construed as misleading information if you're a rat—for the other half. If raising the platform was facilitating re-learning, then animals capable of learning would look for the platform in the new location. But if raising the platform functioned as a reminder, then animals should gravitate toward the place they were trained to, regardless of whether the platform reminder was in the original location or a new one. And that's what happened to the rats with partial lesions.De Hoz et al. then repeated the experiments in a new environment. If the rats managed to escape in this situation, the authors explain, then the reminder treatment could be causing new learning rather than triggering recall of the original training experience. Not surprisingly, the rats with total lesions failed to learn in this new environment (or to be reminded in the previous experiment), while the control rats adapted to the new location. The group with partial lesions failed to learn.Since partially lesioned rats responded to the reminder treatment by recalling the original platform location, their initial memory failure couldn't have resulted from a storage failure. And since they did not realize they could escape from the new location, the reminder didn't induce new learning. Among the many questions these results raise is what role the hippocampus plays in memory storage and retrieval. It could be, as the authors propose, that essential components of spatial memory traces are either stored in the hippocampus or reactivated there, since only the partially lesioned rats responded to reminders. Whether that proves true, de Hoz et al. have contributed a much needed resource for investigating the neural basis of memory loss.


Background There are controversial results on the efficacy of the abandoned, intrathecal predominant methylprednisolone application in multiple sclerosis (MS) in contrast to the proven effectiveness in intractable postherpetic neuralgia.
Background
There are controversial results on the efficacy of the abandoned, intrathecal predominant methylprednisolone application in multiple sclerosis (MS) in contrast to the proven effectiveness in intractable postherpetic neuralgia.Methods We performed an analysis of the efficacy of the application of 40 mg of the sustained release steroid triamcinolone acetonide (TCA). We intrathecally injected in sterile saline dissolved TCA six times within three weeks on a regular basis every third day in 161 hospitalized primary and predominant secondary progressive MS patients with spinal symptoms. The MS patients did not experience an acute onset of exacerbation or recent distinct increased progression of symptoms. We simultaneously scored the MS patients with the EDSS and the Barthel index, estimated the walking distance and measured somatosensory evoked potentials. Additionally the MS patients received a standardized rehabilitation treatment.
Methods
We performed an analysis of the efficacy of the application of 40 mg of the sustained release steroid triamcinolone acetonide (TCA). We intrathecally injected in sterile saline dissolved TCA six times within three weeks on a regular basis every third day in 161 hospitalized primary and predominant secondary progressive MS patients with spinal symptoms. The MS patients did not experience an acute onset of exacerbation or recent distinct increased progression of symptoms. We simultaneously scored the MS patients with the EDSS and the Barthel index, estimated the walking distance and measured somatosensory evoked potentials. Additionally the MS patients received a standardized rehabilitation treatment.Results EDSS score and Barthel index improved, walking distance increased, latencies of somatosensory evoked potentials of the median and tibial nerves shortened in all MS patients with serial evaluation (p < 0.0001 for all variables). Side effects were rare, five patients stopped TCA application due to onset of a post lumbar puncture syndrome.
Results
EDSS score and Barthel index improved, walking distance increased, latencies of somatosensory evoked potentials of the median and tibial nerves shortened in all MS patients with serial evaluation (p < 0.0001 for all variables). Side effects were rare, five patients stopped TCA application due to onset of a post lumbar puncture syndrome.Conclusions Repeated intrathecal TCA application improves spinal symptoms, walking distance and SSEP latencies in progressive MS patients in this uncontrolled study. Future trials should evaluate the long-term benefit of this invasive treatment.
Conclusions
Repeated intrathecal TCA application improves spinal symptoms, walking distance and SSEP latencies in progressive MS patients in this uncontrolled study. Future trials should evaluate the long-term benefit of this invasive treatment.

Background There are controversial results on the efficacy of the nowadays still abandoned, intrathecal steroid application predominantly due to a missing detailed evaluation of patients' characteristics, careful monitoring and standardized outcome measurements in multiple sclerosis (MS) [ 1 ]. Initially, case reports of intrathecal methylprednisolon and ACTH administration described beneficial effects in MS patients, but the following studies showed disappointing results in particular in comparison to systemic steroid application [ 1 ]. Earlier intrathecal treatment trials in MS patients suffered from small sample sizes, low number of injections, low steroid dosages, short half life of the administered steroid and an increasing number of reports on side-effects, i.e. adhesive arachnoiditis and various forms of meninigitis probably due to neurotoxic solvents and bacteriostatic additives [ 4 ]. Moreover a retarded release steroid preparation was not available for many years [ 1 , 5 ]. Then administration of triamcinolone acetonide crystal suspensions (TCA), dissolved at bedside in sterile saline, was introduced in the intrathecal steroid treatment of MS. However, studies again lacked of detailed evaluation and clinical characterization of MS patients, small sample sizes and a low number of intrathecal application of this retarded release steroid compound [ 1 , 4 , 5 ]. Accordingly, there was no convincing superiority over the efficacy of the systemic steroid treatment. Some enrolled MS patients experienced a recent deterioration of symptoms due to prior acute relapses and/or ongoing chronic progression. Moreover study participants were not classified according to the various subtypes of MS progression [ 5 , 6 ]. However in general, a comparison of both methods of steroid application is at least doubtful from the pharmacokinetic point of view. The resulting steroid efficacy in the central nervous system enormously differs in favor for the intraspinal application due to the achieved cerebrospinal fluid steroid level, marked longer half life, i.e. with detection of TCA even four months after the last administration, and missing impact on the endogenous peripheral cortisol secretion with no appearance of side effects of systemic high dosage steroid application [ 4 , 6 ]. In recent years, a certain revival of intrathecal methylprednisolon administration took place in the treatment of intractable postherpetic neuralgia and MS with spinal symptoms, both of which turned out to be very effective, but were controversially discussed regarding the safety issues [ 2 , 3 ]. The MS study demonstrated, that six repeat intrathecal TCA injections within three weeks reduced the EDSS score in 31 of 36 progressive MS patients with predominant spinal symptoms. 20 of them entered a follow-up period of 13.1 ± 6.22, 3 – 23 [mean ± SD, range] months with 6.35 ± 3.91, 2 – 15 TCA injections. They received one TCA application on a regular basis in an individually differing frequency every six to twelve weeks. These patients remained stable [ 3 ]. Nevertheless, there is a need for further results on the usefulness of this treatment. The optimum design would be a placebo-controlled arm, but repeat performance of intrathecal saline (placebo) administration under double-blind conditions with the patients' consent and an approval of an ethical committee is not realistic in clinical practice. Moreover they may be ethical concerns of withholding treatment [ 7 ]. Our present study is a way out of this dilemma of the debate on the efficacy of TCA treatment, which is carried out in certain specific centers in Germany for many years now [ 3 ]. We performed an analysis of standardized intrathecal application of TCA in MS patients with spinal symptoms, using subjective rating procedures and objective measurements.
Background
There are controversial results on the efficacy of the nowadays still abandoned, intrathecal steroid application predominantly due to a missing detailed evaluation of patients' characteristics, careful monitoring and standardized outcome measurements in multiple sclerosis (MS) [ 1 ]. Initially, case reports of intrathecal methylprednisolon and ACTH administration described beneficial effects in MS patients, but the following studies showed disappointing results in particular in comparison to systemic steroid application [ 1 ]. Earlier intrathecal treatment trials in MS patients suffered from small sample sizes, low number of injections, low steroid dosages, short half life of the administered steroid and an increasing number of reports on side-effects, i.e. adhesive arachnoiditis and various forms of meninigitis probably due to neurotoxic solvents and bacteriostatic additives [ 4 ]. Moreover a retarded release steroid preparation was not available for many years [ 1 , 5 ]. Then administration of triamcinolone acetonide crystal suspensions (TCA), dissolved at bedside in sterile saline, was introduced in the intrathecal steroid treatment of MS. However, studies again lacked of detailed evaluation and clinical characterization of MS patients, small sample sizes and a low number of intrathecal application of this retarded release steroid compound [ 1 , 4 , 5 ].Accordingly, there was no convincing superiority over the efficacy of the systemic steroid treatment. Some enrolled MS patients experienced a recent deterioration of symptoms due to prior acute relapses and/or ongoing chronic progression. Moreover study participants were not classified according to the various subtypes of MS progression [ 5 , 6 ]. However in general, a comparison of both methods of steroid application is at least doubtful from the pharmacokinetic point of view. The resulting steroid efficacy in the central nervous system enormously differs in favor for the intraspinal application due to the achieved cerebrospinal fluid steroid level, marked longer half life, i.e. with detection of TCA even four months after the last administration, and missing impact on the endogenous peripheral cortisol secretion with no appearance of side effects of systemic high dosage steroid application [ 4 , 6 ]. In recent years, a certain revival of intrathecal methylprednisolon administration took place in the treatment of intractable postherpetic neuralgia and MS with spinal symptoms, both of which turned out to be very effective, but were controversially discussed regarding the safety issues [ 2 , 3 ]. The MS study demonstrated, that six repeat intrathecal TCA injections within three weeks reduced the EDSS score in 31 of 36 progressive MS patients with predominant spinal symptoms. 20 of them entered a follow-up period of 13.1 ± 6.22, 3 – 23 [mean ± SD, range] months with 6.35 ± 3.91, 2 – 15 TCA injections. They received one TCA application on a regular basis in an individually differing frequency every six to twelve weeks. These patients remained stable [ 3 ]. Nevertheless, there is a need for further results on the usefulness of this treatment. The optimum design would be a placebo-controlled arm, but repeat performance of intrathecal saline (placebo) administration under double-blind conditions with the patients' consent and an approval of an ethical committee is not realistic in clinical practice. Moreover they may be ethical concerns of withholding treatment [ 7 ]. Our present study is a way out of this dilemma of the debate on the efficacy of TCA treatment, which is carried out in certain specific centers in Germany for many years now [ 3 ]. We performed an analysis of standardized intrathecal application of TCA in MS patients with spinal symptoms, using subjective rating procedures and objective measurements.Methods Subjects We only enrolled clinically well characterized, consecutively referred MS patients (table  1 ,  2 ) with distinct spinal symptoms and/or MRT visualized lesions in the spinal cord [ 8 ]. These patients did not suffer from an acute onset of exacerbation or recent clearly increased progression of their symptoms. Table 1 Patients' characteristics. Age 50.10 ± 10.30, 21 – 78 years Duration of MS 14.32 ± 7.63, 2 – 40 years Sex 119 men, 42 women MS types chronic progressive: n = 35 secondary progressive: n = 122 relapsing-remitting: n = 4 Length of hospital stay 28.41 ± 5.97; 21 – 60 days Table 2 Treatment against spasticity. Before TCA After TCA Baclofen 7.56 ± 16.06; 0 – 80 mg, n = 117 without baclofen 6.44 ± 15.51; 0 – 80 mg, n = 125 without baclofen Tolperison 1.19 ± 8.20; 0 – 75 mg, n = 154 without tolperison 1.34 ± 7.41; 0 – 50 mg, n = 152 without tolperison Tizanidin 1.94 ± 5.29; 0 – 32 mg, n = 132 without tizanidin 3.38 ± 6.64; 0 – 32 mg, n = 110 without tizanidin All data are given as mean ± standard deviation; minimum – maximum; n = number of patients; TCA = standardized intrathecal triamcinolon acetonide application according to the methods section. Methods We performed scoring with both, EDSS and Barthel index and assessed the walking distance. Then we measured somatosensory evoked potentials (SSEP) in a standardized fashion before start and at the end of the intraspinal TCA treatment within a prospective study design [ 9 ]. A technician performed SSEP recordings and measured the walking distance. We blinded the EDSS raters. Retrospectively, we compiled information on patients from their hospital records, i.e. date of birth, sex, duration of disease after diagnosis of MS, dosages of oral baclofen (lioresal ® ), tolperison (mydocalm ® ), tizanidin (sirdalud ® ) on the first and last day of the hospital stay, length of hospitalization in days (tables  1  & 2 ). The patients additionally received a standardized rehabilitation treatment, which included physiotherapy, massage and optional swimming with the patients' consent [ 12 , 13 ]. Only data with successfully performed serial evaluation of patients were compared for each variable. We performed lumbar puncture with an "atraumatic" Sprotte needle [ 10 , 11 ]. Each patient received six intrathecal applications of 40 mg TCA followed by a mandatory stay in bed for at least six hours. This should reduce incidence of lumbar puncture syndrome and hypothetically support the diffusion of TCA in the CSF and the spinal cord [ 14 , 15 ]. A preexisting immune system modulating drug therapy remained stable. We closely monitored for typical concomitant with systemic steroid application appearing side effects, i.e. increase of body weight etc., all of which did not significantly change (data not shown) [ 16 ]. No slight or severe side effects occurred, but we did not include five patients into our evaluation due to onset of post lumbar puncture syndrome with headache and nausea, which caused a stop of further intrathecal TCA applications. These patients withdraw their consent. We only considered SSEP data of patients with serial measurements, which we performed on the same day of the EDSS rating. Ethics Each participant gave written informed consent for the TCA treatment, which was approved by the local ethical committee. The consent form included a detailed description of all putative risks of lumbar puncture and intrathecal TCA application. Statistics Data showed a normal distribution according to the Kolmogorow-Smirnow test. As a result, we only performed parametric tests. We used ANCOVA with repeated measures design including MS duration, MS types, change of dosages of concomitant drugs against spasticity, length of hospital stay, sex and age as covariates. We computed SSEP results by adding both sides in order to reduce amount of calculations for comparisons. Then we calculated the differences of latencies between both timepoints of recordings according to the formula [Initial - End = Diff] for correlation analysis. We employed linear regression for correlation analysis. Level of significance of p-values were adjusted to 0.05 divided by the number of performed comparisons respectively correlations.
Methods
Subjects We only enrolled clinically well characterized, consecutively referred MS patients (table  1 ,  2 ) with distinct spinal symptoms and/or MRT visualized lesions in the spinal cord [ 8 ]. These patients did not suffer from an acute onset of exacerbation or recent clearly increased progression of their symptoms. Table 1 Patients' characteristics. Age 50.10 ± 10.30, 21 – 78 years Duration of MS 14.32 ± 7.63, 2 – 40 years Sex 119 men, 42 women MS types chronic progressive: n = 35 secondary progressive: n = 122 relapsing-remitting: n = 4 Length of hospital stay 28.41 ± 5.97; 21 – 60 days Table 2 Treatment against spasticity. Before TCA After TCA Baclofen 7.56 ± 16.06; 0 – 80 mg, n = 117 without baclofen 6.44 ± 15.51; 0 – 80 mg, n = 125 without baclofen Tolperison 1.19 ± 8.20; 0 – 75 mg, n = 154 without tolperison 1.34 ± 7.41; 0 – 50 mg, n = 152 without tolperison Tizanidin 1.94 ± 5.29; 0 – 32 mg, n = 132 without tizanidin 3.38 ± 6.64; 0 – 32 mg, n = 110 without tizanidin All data are given as mean ± standard deviation; minimum – maximum; n = number of patients; TCA = standardized intrathecal triamcinolon acetonide application according to the methods section.
Subjects
We only enrolled clinically well characterized, consecutively referred MS patients (table  1 ,  2 ) with distinct spinal symptoms and/or MRT visualized lesions in the spinal cord [ 8 ]. These patients did not suffer from an acute onset of exacerbation or recent clearly increased progression of their symptoms.Patients' characteristics.Treatment against spasticity.All data are given as mean ± standard deviation; minimum – maximum; n = number of patients; TCA = standardized intrathecal triamcinolon acetonide application according to the methods section.Methods We performed scoring with both, EDSS and Barthel index and assessed the walking distance. Then we measured somatosensory evoked potentials (SSEP) in a standardized fashion before start and at the end of the intraspinal TCA treatment within a prospective study design [ 9 ]. A technician performed SSEP recordings and measured the walking distance. We blinded the EDSS raters. Retrospectively, we compiled information on patients from their hospital records, i.e. date of birth, sex, duration of disease after diagnosis of MS, dosages of oral baclofen (lioresal ® ), tolperison (mydocalm ® ), tizanidin (sirdalud ® ) on the first and last day of the hospital stay, length of hospitalization in days (tables  1  & 2 ). The patients additionally received a standardized rehabilitation treatment, which included physiotherapy, massage and optional swimming with the patients' consent [ 12 , 13 ]. Only data with successfully performed serial evaluation of patients were compared for each variable. We performed lumbar puncture with an "atraumatic" Sprotte needle [ 10 , 11 ]. Each patient received six intrathecal applications of 40 mg TCA followed by a mandatory stay in bed for at least six hours. This should reduce incidence of lumbar puncture syndrome and hypothetically support the diffusion of TCA in the CSF and the spinal cord [ 14 , 15 ]. A preexisting immune system modulating drug therapy remained stable. We closely monitored for typical concomitant with systemic steroid application appearing side effects, i.e. increase of body weight etc., all of which did not significantly change (data not shown) [ 16 ]. No slight or severe side effects occurred, but we did not include five patients into our evaluation due to onset of post lumbar puncture syndrome with headache and nausea, which caused a stop of further intrathecal TCA applications. These patients withdraw their consent. We only considered SSEP data of patients with serial measurements, which we performed on the same day of the EDSS rating.
Methods
We performed scoring with both, EDSS and Barthel index and assessed the walking distance. Then we measured somatosensory evoked potentials (SSEP) in a standardized fashion before start and at the end of the intraspinal TCA treatment within a prospective study design [ 9 ]. A technician performed SSEP recordings and measured the walking distance. We blinded the EDSS raters. Retrospectively, we compiled information on patients from their hospital records, i.e. date of birth, sex, duration of disease after diagnosis of MS, dosages of oral baclofen (lioresal ® ), tolperison (mydocalm ® ), tizanidin (sirdalud ® ) on the first and last day of the hospital stay, length of hospitalization in days (tables  1  & 2 ). The patients additionally received a standardized rehabilitation treatment, which included physiotherapy, massage and optional swimming with the patients' consent [ 12 , 13 ]. Only data with successfully performed serial evaluation of patients were compared for each variable. We performed lumbar puncture with an "atraumatic" Sprotte needle [ 10 , 11 ]. Each patient received six intrathecal applications of 40 mg TCA followed by a mandatory stay in bed for at least six hours. This should reduce incidence of lumbar puncture syndrome and hypothetically support the diffusion of TCA in the CSF and the spinal cord [ 14 , 15 ]. A preexisting immune system modulating drug therapy remained stable. We closely monitored for typical concomitant with systemic steroid application appearing side effects, i.e. increase of body weight etc., all of which did not significantly change (data not shown) [ 16 ]. No slight or severe side effects occurred, but we did not include five patients into our evaluation due to onset of post lumbar puncture syndrome with headache and nausea, which caused a stop of further intrathecal TCA applications. These patients withdraw their consent. We only considered SSEP data of patients with serial measurements, which we performed on the same day of the EDSS rating.Ethics Each participant gave written informed consent for the TCA treatment, which was approved by the local ethical committee. The consent form included a detailed description of all putative risks of lumbar puncture and intrathecal TCA application.
Ethics
Each participant gave written informed consent for the TCA treatment, which was approved by the local ethical committee. The consent form included a detailed description of all putative risks of lumbar puncture and intrathecal TCA application.Statistics Data showed a normal distribution according to the Kolmogorow-Smirnow test. As a result, we only performed parametric tests. We used ANCOVA with repeated measures design including MS duration, MS types, change of dosages of concomitant drugs against spasticity, length of hospital stay, sex and age as covariates. We computed SSEP results by adding both sides in order to reduce amount of calculations for comparisons. Then we calculated the differences of latencies between both timepoints of recordings according to the formula [Initial - End = Diff] for correlation analysis. We employed linear regression for correlation analysis. Level of significance of p-values were adjusted to 0.05 divided by the number of performed comparisons respectively correlations.
Statistics
Data showed a normal distribution according to the Kolmogorow-Smirnow test. As a result, we only performed parametric tests. We used ANCOVA with repeated measures design including MS duration, MS types, change of dosages of concomitant drugs against spasticity, length of hospital stay, sex and age as covariates. We computed SSEP results by adding both sides in order to reduce amount of calculations for comparisons. Then we calculated the differences of latencies between both timepoints of recordings according to the formula [Initial - End = Diff] for correlation analysis. We employed linear regression for correlation analysis. Level of significance of p-values were adjusted to 0.05 divided by the number of performed comparisons respectively correlations.Results Comparisons EDSS score (n [number of subjects with serial evaluation] = 161) and Barthel index (n = 68) improved and walking distance (n = 161) increased (table  3 ). SSEP latencies of tibial (n = 136) and median (n = 108) nerves reduced (table  3 ). P-values of all performed comparisons were below 0.0001. No significant effects of covariates appeared. Table 3 Comparison of clinical data. Before TCA After TCA F EDSS – score 6.44 ± 1.06; 3.5 – 6.5 5.47 ± 1.24; 2 – 8.5 379.28 walking distance 158.03 ± 501.20; 0 – 5000 439.38 ± 895.24; 0 – 5000 34.40 Barthel index 58 ± 20.07; 5 – 100 89.13 ± 12.57; 60 – 100 347.52 P2 (tibial nerve) 105.80 ± 10.38; 85 – 130 88.57 ± 5.60; 77 – 106 470.96 N2 (tibial nerve) 118.83 ± 10.49; 92 – 148 101.63 ± 6.43; 80 – 118 425.71 P3 (tibial nerve) 131.79 ± 13.22; 70 – 164 114.31 ± 7.46; 95 – 135 325.88 N2 (median nerve) 46.53 ± 5.40; 23–60 41.03 ± 2.92; 23–47 138.10 P2 (median nerve) 52.73 ± 6.34; 25 – 68 47.09 ± 3.03; 41–56 128.80 All data are given as mean ± standard deviation; minimum – maximum in the second and third column; latencies (N2, P2, P3) of the somatosensensory evoked potentials are given in milliseconds, walking distance is given in meters, F = F-value of ANCOVA, SD = standard deviation; TCA = standardized intrathecal triamcinolon acetonide application according to the methods section. Correlation analysis There were no significant relations between computed changes of EDSS scores, walking distances and SSEP results (results not shown). Diff P2 correlated with Diff P3 (R [correlation coefficient] = 0.71), Diff P2 with Diff N2 (R = 0.90), Diff P3 with Diff N2 (R = 0.74) of SSEP latencies of the tibial nerves. There were relations between Diff N2 and Diff P2 (R = 0.85) of SSEP latencies of the median nerves. P-values of these correlations were below 0.0001. Moreover Diff P2 of the tibial nerves correlated with Diff N2 of the median nerves (R = 0.28, p = 0.004, n = 100). There was a certain trend for a significant correlation between Diff P2 of the tibial nerve and Diff P2 of the median nerve (R = 0.22, p = 0.03). No other significant associations of SSEP data appeared (results not shown).
Results
Comparisons EDSS score (n [number of subjects with serial evaluation] = 161) and Barthel index (n = 68) improved and walking distance (n = 161) increased (table  3 ). SSEP latencies of tibial (n = 136) and median (n = 108) nerves reduced (table  3 ). P-values of all performed comparisons were below 0.0001. No significant effects of covariates appeared. Table 3 Comparison of clinical data. Before TCA After TCA F EDSS – score 6.44 ± 1.06; 3.5 – 6.5 5.47 ± 1.24; 2 – 8.5 379.28 walking distance 158.03 ± 501.20; 0 – 5000 439.38 ± 895.24; 0 – 5000 34.40 Barthel index 58 ± 20.07; 5 – 100 89.13 ± 12.57; 60 – 100 347.52 P2 (tibial nerve) 105.80 ± 10.38; 85 – 130 88.57 ± 5.60; 77 – 106 470.96 N2 (tibial nerve) 118.83 ± 10.49; 92 – 148 101.63 ± 6.43; 80 – 118 425.71 P3 (tibial nerve) 131.79 ± 13.22; 70 – 164 114.31 ± 7.46; 95 – 135 325.88 N2 (median nerve) 46.53 ± 5.40; 23–60 41.03 ± 2.92; 23–47 138.10 P2 (median nerve) 52.73 ± 6.34; 25 – 68 47.09 ± 3.03; 41–56 128.80 All data are given as mean ± standard deviation; minimum – maximum in the second and third column; latencies (N2, P2, P3) of the somatosensensory evoked potentials are given in milliseconds, walking distance is given in meters, F = F-value of ANCOVA, SD = standard deviation; TCA = standardized intrathecal triamcinolon acetonide application according to the methods section.
Comparisons
EDSS score (n [number of subjects with serial evaluation] = 161) and Barthel index (n = 68) improved and walking distance (n = 161) increased (table  3 ). SSEP latencies of tibial (n = 136) and median (n = 108) nerves reduced (table  3 ). P-values of all performed comparisons were below 0.0001. No significant effects of covariates appeared.Comparison of clinical data.All data are given as mean ± standard deviation; minimum – maximum in the second and third column; latencies (N2, P2, P3) of the somatosensensory evoked potentials are given in milliseconds, walking distance is given in meters, F = F-value of ANCOVA, SD = standard deviation; TCA = standardized intrathecal triamcinolon acetonide application according to the methods section.Correlation analysis There were no significant relations between computed changes of EDSS scores, walking distances and SSEP results (results not shown). Diff P2 correlated with Diff P3 (R [correlation coefficient] = 0.71), Diff P2 with Diff N2 (R = 0.90), Diff P3 with Diff N2 (R = 0.74) of SSEP latencies of the tibial nerves. There were relations between Diff N2 and Diff P2 (R = 0.85) of SSEP latencies of the median nerves. P-values of these correlations were below 0.0001. Moreover Diff P2 of the tibial nerves correlated with Diff N2 of the median nerves (R = 0.28, p = 0.004, n = 100). There was a certain trend for a significant correlation between Diff P2 of the tibial nerve and Diff P2 of the median nerve (R = 0.22, p = 0.03). No other significant associations of SSEP data appeared (results not shown).
Correlation analysis
There were no significant relations between computed changes of EDSS scores, walking distances and SSEP results (results not shown). Diff P2 correlated with Diff P3 (R [correlation coefficient] = 0.71), Diff P2 with Diff N2 (R = 0.90), Diff P3 with Diff N2 (R = 0.74) of SSEP latencies of the tibial nerves. There were relations between Diff N2 and Diff P2 (R = 0.85) of SSEP latencies of the median nerves. P-values of these correlations were below 0.0001. Moreover Diff P2 of the tibial nerves correlated with Diff N2 of the median nerves (R = 0.28, p = 0.004, n = 100). There was a certain trend for a significant correlation between Diff P2 of the tibial nerve and Diff P2 of the median nerve (R = 0.22, p = 0.03). No other significant associations of SSEP data appeared (results not shown).Discussion Our results demonstrate and confirm the efficacy of repeated intraspinal TCA application in MS patients with spinal symptoms, which improved according to the EDSS outcomes and the results of assessed walking distance and determined SSEP latencies [ 3 ]. We intrathecally injected TCA six times within three weeks, whereas earlier trials weekly performed one application up to three times at the most [ 4 ]. The distinct reduction of SSEP peak latencies and the significant relations between their computed differences confirm the clinical outcomes and underline the efficacy of intraspinal TCA treatment in MS patients with spinal symptoms in general. We hypothesize, that these neurophysiological results indicate a certain remyelinating and/or restorative potential of intraspinal TCA application with an at least transient shift from chronic inflammation to remyelination [ 17 , 18 ]. Our results support the crucially discussed view, that serial SSEP studies in MS may monitor the effect of treatment to a certain extent under standardized conditions [ 9 , 19 , 20 ]. Our analysis also shows that primary and secondary progressive even advanced MS patients with spinal symptoms predominantly improve from this kind of intrathecal steroid therapy. We assume, that we achieve persistent high steroid concentrations at lesions of the spinal cord, since TCA must not pass the blood brain barrier [ 6 ]. However previous comparisons of the clinical efficacy of intrathecal TCA application with the intravenous administration of methylprednisolone showed no superiority of one method over the other [ 5 , 6 , 19 ]. But these studies did not exclude relapsing remitting patients or participants with a previous acute relapse. They did not focus on spinal symptoms. Their application rate of TCA was distinct lower compared with the one of our present and a previous trial [ 3 ]. However our present study outcomes do not allow any conclusions on the duration of the achieved benefit and the impact of TCA treatment on progression of MS [ 3 ]. Therefore there is an urgent need for further confirmatory trials, which additionally address all these issues. A strategy would be to choose one arm with active treatment and one arm with just follow-up without active treatment with blind assessment by an evaluating physician. However we stress concerning long-term steroid therapy and progression of MS, that there are positive outcomes of trials with intravenous methylprednisolone administration in various application rates and dosages on long term disease progression and/or on brain atrophy in secondary-progressive -, respectively relapsing-remitting MS patients [ 16 , 21 ]. In contrast to studies on intravenous oral steroid treatment, we did not observe the typical side effects of systemic high dosage steroid administration, i.e. edema. This may support previous findings by circumstantial evidence, which report no decrease of endogenous cortisol secretion following intrathecal TCA administration [ 4 ]. We cannot exclude a certain impact of physiotherapy, the standardized rehabilitation treatment and an beneficial effect of hospitalization in general with its resulting concomitant positive influence on activities of daily living [ 12 , 13 ]. However, we found no significant impact of the corresponding covariate length of the hospital stay in our statistical analysis. We assume, that our results do not reflect an improved drug therapy against spasticity, since no significant impact of the covariate computed changes of medication appeared. Nevertheless we cannot exclude a certain effect of the steroid on spasticity. However most participants did not take any drug against spasticity. Onset of side effects of lumbar puncture itself were negligible, since we used an atraumatic needle [ 11 ].
Discussion
Our results demonstrate and confirm the efficacy of repeated intraspinal TCA application in MS patients with spinal symptoms, which improved according to the EDSS outcomes and the results of assessed walking distance and determined SSEP latencies [ 3 ]. We intrathecally injected TCA six times within three weeks, whereas earlier trials weekly performed one application up to three times at the most [ 4 ]. The distinct reduction of SSEP peak latencies and the significant relations between their computed differences confirm the clinical outcomes and underline the efficacy of intraspinal TCA treatment in MS patients with spinal symptoms in general. We hypothesize, that these neurophysiological results indicate a certain remyelinating and/or restorative potential of intraspinal TCA application with an at least transient shift from chronic inflammation to remyelination [ 17 , 18 ]. Our results support the crucially discussed view, that serial SSEP studies in MS may monitor the effect of treatment to a certain extent under standardized conditions [ 9 , 19 , 20 ]. Our analysis also shows that primary and secondary progressive even advanced MS patients with spinal symptoms predominantly improve from this kind of intrathecal steroid therapy. We assume, that we achieve persistent high steroid concentrations at lesions of the spinal cord, since TCA must not pass the blood brain barrier [ 6 ]. However previous comparisons of the clinical efficacy of intrathecal TCA application with the intravenous administration of methylprednisolone showed no superiority of one method over the other [ 5 , 6 , 19 ]. But these studies did not exclude relapsing remitting patients or participants with a previous acute relapse. They did not focus on spinal symptoms. Their application rate of TCA was distinct lower compared with the one of our present and a previous trial [ 3 ].However our present study outcomes do not allow any conclusions on the duration of the achieved benefit and the impact of TCA treatment on progression of MS [ 3 ]. Therefore there is an urgent need for further confirmatory trials, which additionally address all these issues. A strategy would be to choose one arm with active treatment and one arm with just follow-up without active treatment with blind assessment by an evaluating physician. However we stress concerning long-term steroid therapy and progression of MS, that there are positive outcomes of trials with intravenous methylprednisolone administration in various application rates and dosages on long term disease progression and/or on brain atrophy in secondary-progressive -, respectively relapsing-remitting MS patients [ 16 , 21 ]. In contrast to studies on intravenous oral steroid treatment, we did not observe the typical side effects of systemic high dosage steroid administration, i.e. edema. This may support previous findings by circumstantial evidence, which report no decrease of endogenous cortisol secretion following intrathecal TCA administration [ 4 ].We cannot exclude a certain impact of physiotherapy, the standardized rehabilitation treatment and an beneficial effect of hospitalization in general with its resulting concomitant positive influence on activities of daily living [ 12 , 13 ]. However, we found no significant impact of the corresponding covariate length of the hospital stay in our statistical analysis. We assume, that our results do not reflect an improved drug therapy against spasticity, since no significant impact of the covariate computed changes of medication appeared. Nevertheless we cannot exclude a certain effect of the steroid on spasticity. However most participants did not take any drug against spasticity. Onset of side effects of lumbar puncture itself were negligible, since we used an atraumatic needle [ 11 ].Conclusions Our data demonstrate the efficacy and safety of repeated intrathecal TCA application in MS patients with predominant spinal symptoms, which markedly improved. Some MS patients experienced post lumbar puncture syndrome with a frequency within the normal range [ 11 ], but typical side effects of systemic high dosage steroid administration did not appear.
Conclusions
Our data demonstrate the efficacy and safety of repeated intrathecal TCA application in MS patients with predominant spinal symptoms, which markedly improved. Some MS patients experienced post lumbar puncture syndrome with a frequency within the normal range [ 11 ], but typical side effects of systemic high dosage steroid administration did not appear.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions KH, FS, HP and TM designed, coordinated and carried out the study. TM performed statistical data analysis and drafted the manuscript. All authors read and approved the final manuscript.
Authors' contributions
KH, FS, HP and TM designed, coordinated and carried out the study. TM performed statistical data analysis and drafted the manuscript. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: