Organ Weights in NPC1 Mutant Mice Partly Normalized by Various Pharmacological Treatment Approaches

Niemann-Pick Type C1 (NPC1, MIM 257220) is a rare, progressive, lethal, inherited autosomal-recessive endolysosomal storage disease caused by mutations in the NPC1 leading to intracellular lipid storage. We analyzed mostly not jet known alterations of the weights of 14 different organs in the BALB/cNctr-Npc1m1N/-J Jackson Npc1 mice in female and male Npc1+/+ and Npc1−/− mice under various treatment strategies. Mice were treated with (i) no therapy, (ii) vehicle injection, (iii) a combination of miglustat, allopregnanolone, and 2-hydroxypropyl-ß-cyclodextrin (HPßCD), (iv) miglustat, and (v) HPßCD alone starting at P7 and repeated weekly throughout life. The 12 respective male and female wild-type mice groups were evaluated in parallel. In total, 351 mice (176 Npc1+/+, 175 Npc1−/−) were dissected at P65. In both sexes, the body weights of None and Sham Npc1−/− mice were lower than those of respective Npc1+/+ mice. The influence of the Npc1 mutation and/or sex on the weights of various organs, however, differed considerably. In males, Npc1+/+ and Npc1−/− mice had comparable absolute weights of lungs, spleen, and adrenal glands. In Npc1−/− mice, smaller weights of hearts, livers, kidneys, testes, vesicular, and scent glands were found. In female Npc1−/− mice, ovaries, and uteri were significantly smaller. In Npc1−/− mice, relative organ weights, i.e., normalized with body weights, were sex-specifically altered to different extents by the different therapies. The combination of miglustat, allopregnanolone, and the sterol chelator HPßCD partly normalized the weights of more organs than miglustat or HPßCD mono-therapies.

According to Ramirez et al. [68], weekly administration of HPßCD overcomes the lysosomal transport defect associated with the Npc1 mutation, nearly normalizes hepatic and whole-animal cholesterol pools, prevents the development of liver disease and slows down cerebellar neurodegeneration, but has little or no effect on the development of progressive pulmonary disease. HPßCD administration reverses the cholesterol transport defect seen in the Npc1 −/− mice at any age, and this reversal allows the sequestered sterol to be excreted from the body as bile acid [4]. In addition, cyclic oligosaccharides are known to extract cholesterol from the plasma membrane of a variety of cells in vitro [69][70][71]. Therefore, it seems clear that not ALLO but HPßCD alone was responsible for most and possibly for all of the effects of ALLO/HPßCD treatment [59,60,63,64].
NPC1 disease is one of many lysosomal storage diseases and results mainly from a mutation that inactivates the NPC1 protein responsible for the transport of unesterified cholesterol from the late endosomal/lysosomal compartment to the cytosol in every cell [76]. This causes cholesterol to accumulate in all organs and practically in all tissues in the body; the accumulation is characterized by progressive enlargement of the internal organs, causing organ dysfunction, which can manifest clinically as hepatosplenomegaly, splenomegaly, prolonged newborn jaundice, liver dysfunction, lipoid pneumonitis, lung failure and, ultimately, progressive neurological dysfunction secondary to selective neurodegeneration [28,54,68,[77][78][79][80][81][82].
With the exception of a recent study [75], most studies on the therapeutic effects of various drugs in Npc1 −/− mice and the respective wild-types were evaluated in gendermixed groups [37,40,59,73,[83][84][85]. Also, clinical observations in NPC1 patients described primarily only age-dependent heterogeneity of the beginning, expression, and symptoms of the disease without differentiation of the patients' gender [86][87][88][89]. Only the study performed by Walterfang et al. [90] described two siblings with schizophrenia whose adult NPC genotypes were identical but showed dimorphism in their illness courses and their clinical and biochemical parameters due to gender. The authors suggest that the course of the disease and the degree of impairment may be different in female patients and that sex hormones may play a role. However, they stated that human data on the effect of sex on the biochemical and clinical parameters in NPC disease are lacking.
Recently, using a mouse model of NPC1 [75], the therapeutic effects of the COMBI therapy with that of MIGLU or HPßCD alone on body and brain weight and the behavior of Npc1 −/− mice were compared in a larger cohort, with special reference to gender differences. Our results suggest that, in Npc1 −/− mice, each drug treatment regimen had a beneficial effect on at least some of the parameters evaluated when compared to Sham-treated mice, partly showing gender-specific therapeutical benefit.
Although in NPC1 lysosomal storage of various lipids is found throughout the body, until now, the existing few studies in different animal models of NPC1 described only the alterations of some disease-involved organs, including mainly liver, spleen, lung, and brain [28,54,84,[91][92][93][94][95][96][97][98] (Supplementary Materials Table S1). Therefore, in the present study, we analyzed, for the first time, alterations of weight and relative weight, dimension, and volume of 14 organs in BALB/cNctr-Npc1 m1N /-J Jackson Npc1 mouse strain in larger cohorts of female and male Npc1 +/+ and Npc1 −/− mice. All parameters were evaluated in one trial after COMBI medication as well as after exclusive MIGLU and HPßCD medication in the different groups, each consisting of more than 12 animals and with special reference to gender: male and female Npc1 −/− mutant mice treated with (i) no therapy (None), (ii) vehicle injection (Sham), (iii) combination of MIGLU, ALLO, and HPßCD (COMBI), (iv) MIGLU alone (MIGLU), (v) HPßCD alone starting at P7 and repeated weekly through-out life (HPßCD), and (vi) HPßCD alone given only once at P7 (HPßCD1x). The 12 respective Npc1 +/+ mouse (male and female wild-type mice) groups were evaluated in parallel. In addition to the recently evaluated mice (n = 239; [75]), further mice (n = 112) from ongoing experiments were included here. Thus, this study is based on the dissection of 351 mice (176 Npc1 +/+ , 175 Npc1 −/− ) evaluated in the 24 groups. A total of 6835 organ measurements were included in the study.
Because we did not find noticeable differences in the weights between the left and right sides in any pair of organs in male and female animals, we further analyzed averaged weight values.
In all mice of the 12 female Npc1 −/− groups, the relative heart weights were in a similar range between 0.485 and 0.577% of body weight ( Figure 2D).

Lungs (Both Sides)
Left and right lungs were weighed together because of their known side-specific anatomical differences in the mouse [99][100][101].
Male mice: The lung weights of male Npc1 +/+ mice of all six groups were in the range of 0.414 to 0.554 g and did not differ significantly between groups ( Figure 3A). In male Npc1 −/− mice, weights were comparable (range: 0.388 to 0.542 g). Only the lung weights of the COMBI group (0.516 ± 0.030 g) significantly surpassed the values of the None group (0.388 ± 0.026 g) (p = 0.028) ( Figure 3A). The lung weights of the male Npc1 +/+ and Npc1 −/− mice in the respective experimental groups did not differ significantly.  The relative lung weights of the male Npc1 +/+ mice of all groups were in the range of 1.75 to 2.20% and did not differ significantly between the treatment groups ( Figure 3C). The same holds true for the relative lung weights of the male Npc1 −/− mice, except for larger values in the Sham group ( Figure 3C). The relative lung weights of the male Npc1 −/− mice generally exceeded those of the respective group of Npc1 +/+ mice, reaching a significant The relative lung weights of the male Npc1 +/+ mice of all groups were in the range of 1.75 to 2.20% and did not differ significantly between the treatment groups ( Figure 3C). The same holds true for the relative lung weights of the male Npc1 −/− mice, except for larger values in the Sham group ( Figure 3C). The relative lung weights of the male Npc1 −/− mice generally exceeded those of the respective group of Npc1 +/+ mice, reaching a significant level in the None, Sham, MIGLU, and HPßCD groups.
The relative lung weights of all female Npc1 +/+ mice were in the range of 1.93 to 2.39%, without significant differences between the respective treatment groups ( Figure 3D). Also, no significant intergroup differences were found in female Npc1 −/− mice ( Figure 3D).
Although the relative lung weights in all-female groups tentatively exceeded the respective values of the Npc1 +/+ mice, significantly increased relative lung weights were only found in the None and HPßCDx1 groups ( Figure 3D).

Liver
Male mice: The liver weights of the male Npc1 +/+ mice of groups None (1.549 ± 0.0699 g) and Sham (1.570 ± 0.0843 g) did not differ significantly ( Figure 4A). The weights of the groups COMBI (1.469 ± 0.0678 g), MIGLU (1.752 ± 0.0932 g), HPßCD (1.457 ± 0.0884 g), and HPßCD1x (1.534 ± 0.140 g) also were not significantly different from the values of the groups None and Sham ( Figure 4A). The liver weights of the male Npc1 −/− mice in groups None (1.312 ± 0.0559 g) and Sham (1.327 ± 0.0932 g) were in the same range, as were the weights of groups HPßCD (1.296 ± 0.106 g) and HPßCD1x (1.540 ± 0.161 g) ( Figure 4A). The liver weights of the None or Sham group were significantly exceeded by the COMBI (1.580 ± 0.0699 g) and the MIGLU (1.796 ± 0.0989 g) groups ( Figure 4A). A comparison of the respective treatment groups revealed that only the Npc1 −/− None group had significantly smaller livers than the wild-type mice ( Figure 4A).
Female mice: The spleen weights of the female Npc1 +/+ and Npc1 −/− mice of all groups did not show any significant differences, probably due to the unexpectedly high interindividual variability, only seen in this organ of female mice ( Figure 5B). Likewise, the relative spleen weights of the Npc1 +/+ and Npc1 −/− mice of all groups did not show significant differences ( Figure 5D).
Female mice: The spleen weights of the female Npc1 +/+ and Npc1 −/− mice of all groups did not show any significant differences, probably due to the unexpectedly high interindividual variability, only seen in this organ of female mice ( Figure 5B). Likewise, the relative spleen weights of the Npc1 +/+ and Npc1 −/− mice of all groups did not show significant differences ( Figure 5D). Male mice: The kidney weights of the male Npc1 +/+ mice of all groups did not show significant differences: None (0.245 ± 0.0084 g), Sham (0.246 ± 0.0094 g), COMBI (0.235 ± 0.0079 g), MIGLU (0.255 ± 0.0103 g), HPßCD (0.213 ± 0.0099 g), and HPßCD1x (0.229 ± 0.0163 g) ( Figure 6A). In the male Npc1 −/− mice, kidney weights of groups None (0.153 ± 0.0064 g) and Sham (0.162 ± 0.0091 g) did not differ significantly ( Figure 6A). The weights of the groups MIGLU (0.177 ± 0.0115 g) and HPßCD1x (0.160 ± 0.0146 g) showed no significant differences from the values of the groups None and Sham. However, the weights of the COMBI (0.210 ± 0075 g) and HPßCD (0.195 ± 0.0115 g) groups significantly exceeded the values of the None group ( Figure 6A). With the exception of the HPßCD group, the kidney weights of all male Npc1 −/− mice groups were significantly lower than those of the comparable Npc1 +/+ groups ( Figure 6A). the values of the None group ( Figure 6A). With the exception of the HPßCD group, the kidney weights of all male Npc1 −/− mice groups were significantly lower than those of the comparable Npc1 +/+ groups ( Figure 6A).
Female mice: There were considerable differences in the kidney weights of the female Npc1 +/+ mice between different groups. Kidneys of the HPßCD1x group (0.137 ± 0.0053 g) were significantly smaller compared with the None (0.167 ± 0.0036 g), Sham (0.166 ± 0.0068 g), and COMBI (0.165 ± 0.0045 g) groups ( Figure 6B), but did not differ significantly from the MIGLU (0.150 ± 0.0064 g) and HPßCD (0.156 ± 0.0068 g) groups ( Figure 6B). The kidney weights of the female Npc1 −/− mice in the groups None (0.115 ± 0.0045 g) and Sham (0.133 ± 0.0059 g) were in the range of the MIGLU (0.131 ± 0.0059 g) and HPßCD1x (0.114 ± 0.0051 g) groups ( Figure 6B). With the exception of the COMBI and HPßCD groups, the kidney weights of the other female Npc1 −/− mouse groups were significantly lower than those of the comparable Npc1 +/+ groups ( Figure 6A).
The relative weights of the Npc1 +/+ mice of the None group (0.098 ± 0.007%) fell significantly below the values of groups HPßCD (0.155 ± 0.014%) and HPßCD1x (0.144 ± 0.012%) ( Figure 8D). The relative weights of groups Sham (0.136 ± 0.014 g), COMBI (0.127 ± 0.010 g), and MIGLU (0.146 ± 0.016 g) did not show significant differences from the values the group None ( Figure 8D). The relative bladder weight of the Npc1 −/− mice of the MIGLU and HPßCD groups was significantly lower than in the respective wild-type groups ( Figure 8D).

Discussion
The organ weights of 175 Npc1 −/− and 176 Npc1 +/+ mice are compared and discussed first with respect to gender and second with respect to the potential benefit of the therapeutic regimens used.
Systematical studies dealing with gender-specific organ weights of Npc1 +/+ and Npc1 −/− mice are rare. With the exception of Xie et al. [102], who found that in both Npc1 +/+ and Npc1 −/− mice, females had lower liver weights than males, all other studies with weight data, to our knowledge, examined gender-mixed groups in a different number of organs.

Npc1 +/+ Control Wild-Type Mice Showed Normal Mouse Organ Weights
The organ weights of the Npc1 +/+ wild-type mice lie in a range comparable with the organ weights dealt with for BALB/C mice [63,84,96,98,106] (Supplementary Materials  Table S1).
Only Xie et al. [102] described the absolute liver weight for both genders separately and reported that the absolute liver weight in male Npc1 +/+ mice was 1.32 ± 0.04 g and in female Npc1 +/+ mice was 1.11 ± 0.03 g. The authors also determined the body weights in both genders separately: the body weight in male Npc1 +/+ mice was 23.1 ± 0.3 and in female Npc1 +/+ mice, 19.6 ± 0.2 g. These data correspond well with our results in female and male Npc1 +/+ mice: the absolute liver weight in male Npc1 +/+ mice was 1.549 ± 0.070 g and in female Npc1 +/+ mice was 1.160 ± 0.038 g. The respective body weight in male Npc1 +/+ mice was 26.601 ± 0.535 and in female Npc1 +/+ mice was 20.558 ± 0.359 g.
Interestingly, we confirm literature data on gender-specific differences in the absolute weight of the adrenal gland. As mentioned by Hedrich [117], we also found higher weights in females compared with males (p < 0.001, Table 1).

Absolute Organ Weights of Npc1 +/+ and Npc1 −/− Mice Mostly Differed in Both Genders
Compared with male Npc1 +/+ mice, the organ weights of the heart, liver, kidney, bladder, testis, vesicular, and scent glands of male Npc1 −/− mice were lower, and femur length was shorter (Table 1). Furthermore, Xie et al. [102] found that the absolute liver weight of male Npc1 +/+ mice at 1.32 ± 0.04 g was lower than that of male Npc1 −/− mice at 1.39 ± 0.04 g. Similarly, the absolute liver weight of the female Npc1 +/+ was lower than that of the Npc1 −/− mice at 1.11 ± 0.03 g and 1.26 ± 0.05 g respectively. No significant differences were found in the weights of the spleen, adrenal gland, or in stomach volumes. Somewhat differing data were found in females: heart, lung, kidney, adrenal gland, ovary, and uterus of the female Npc1 −/− mice had lower weights, and femur length was shorter ( Table 1). No significant differences were found in the weights of the liver, spleen, or in stomach volumes (Table 1).
Seemingly, organ weights of the None mice groups can be roughly divided into two groups: (i) smaller organs were found in smaller mice, or (ii) organ weights were comparable, although mice differed considerably in body weight.
As shown in Figure 15, irrespective of gender and genotype, smaller organ weights were found in the heart ( Figure 15A), kidney ( Figure 15B), bladder ( Figure 15C), testis ( Figure 15D), vesicular gland ( Figure 15E), scent gland ( Figure 15A), ovary ( Figure 15A), and uterus ( Figure 15H) in mice with smaller body weights. It can be assumed that during the specific development of the heart, kidney, and bladder, changed cholesterol metabolism played a subordinate role. Interestingly, hormone-producing and hormone-dependent organs were massively underdeveloped in both genders of Npc1 −/− mice ( Figure 15D-H).
The findings indicate that the infertility of Npc1 −/− mice are reflected in the morphology. Steroid hormones are vital bioactive metabolites derived from cholesterol synthesized in the endoplasmic reticulum and mitochondria [118][119][120], which are divided according to their function and structure into glucocorticoids, mineralocorticoids, estrogens, progestins, androgens, and neurosteroids [121,122]. Because of mitochondrial abnormalities associated with NPC, an abnormal steroid hormone metabolism may be expected [59,[123][124][125][126][127][128][129][130]. The Npc1 −/− gene is important for the normal development of reproductive functions, illustrated by the fact that both Npc1 −/− affected males and females are sterile and have important histological abnormalities in the gonads [125,128]. Gévry et al. [128] showed that female BALB/c npcnih−/− mice are infertile with underdeveloped ovarian follicles, reduced steroidogenesis, no ovulation, and no corpora lutea. The results of this study provided strong evidence for the view that infertility in the BALB/c npcnih−/− mice is attributed to a maturation failure of the ovarian follicle to the great antrum and preovulatory stages. This leads to disruption of the cascade of ovarian and pituitary hormone secretion and prevents normal heat cycles.
Mice with a spontaneous mutation in the Npc1 have been described as infertile [131]. The absence of the functional Npc1 causes abnormalities in spermatogenesis and deregulation of cholesterol homeostasis in the seminiferous tubules. Reducing cholesterol levels is crucial for normal sperm function [132], suggesting that balanced cholesterol levels of the sperm membranes are required for male fertility. The decrease in testosterone synthesis in Npc1 −/− mice suggests that the disrupted cholesterol trafficking in NPC might also disrupt neurosteroidogenesis [59].
The work of Akpovi et al. [125] extends the findings from other studies that reported decreased testosterone production by Leydig cells in Npc1 −/− mice [131]. This was seemingly not due to insufficient precursor availability of cholesterol [123] but to reduced stimulation of the pituitary gland [133], which was corrected by the expression of Npc1 in the glia [134]. Abe et al. [127] analyzed the metabolic changes of steroid hormones in the NPC model and wild-type cells and developed a simultaneous steroid hormone analysis method using LC-MS/MS, which allows for a deeper understanding of NPC pathophysiology and their involvement in mitochondrial steroid hormone production. It was found that testosterone, androsterone, progesterone, and estrone levels were significantly reduced in the NPC model cells [127].  The organ weights of the lung, liver, and spleen were in a comparable range, irrespective of mice gender and Npc1 gene expression ( Figure 16A-C). Our data corroborate published measures (Supplementary Materials Table S1).
ulation of the pituitary gland [133], which was corrected by the expression of Npc1 in the glia [134]. Abe et al. [127] analyzed the metabolic changes of steroid hormones in the NPC model and wild-type cells and developed a simultaneous steroid hormone analysis method using LC-MS/MS, which allows for a deeper understanding of NPC pathophysiology and their involvement in mitochondrial steroid hormone production. It was found that testosterone, androsterone, progesterone, and estrone levels were significantly reduced in the NPC model cells [127].
The organ weights of the lung, liver, and spleen were in a comparable range, irrespective of mice gender and Npc1 gene expression ( Figure 16A-C). Our data corroborate published measures (Supplementary Materials Table S1). Systematic studies dealing with gender-specific organ weights of Npc1 +/+ and Npc1 −/− mice are rare. To our knowledge, only Xie et al. [102] studied both genders separately, stating that in both Npc1 +/+ and Npc1 −/− mice, females had lower liver weights than males. The absolute liver weight was 1.32 ± 0.04 g in male Npc1 +/+ mice and 1.11 ± 0.03 g in female Npc1 +/+ mice. The respective body weights were 23.1 ± 0.3 g in male Npc1 +/+ mice and 19.6 ± 0.2 g in female Npc1 +/+ mice. These data correspond well with our results in female and Systematic studies dealing with gender-specific organ weights of Npc1 +/+ and Npc1 −/− mice are rare. To our knowledge, only Xie et al. [102] studied both genders separately, stating that in both Npc1 +/+ and Npc1 −/− mice, females had lower liver weights than males. The absolute liver weight was 1.32 ± 0.04 g in male Npc1 +/+ mice and 1.11 ± 0.03 g in female Npc1 +/+ mice. The respective body weights were 23.1 ± 0.3 g in male Npc1 +/+ mice and 19.6 ± 0.2 g in female Npc1 +/+ mice. These data correspond well with our results in female and male Npc1 +/+ mice: the absolute liver weight in male Npc1 +/+ mice was 1.549 ± 0.070 g and in female Npc1 +/+ mice, 1.160 ± 0.038 g. The respective body weight in male Npc1 +/+ mice was 26.601 ± 0.535 g and in female Npc1 +/+ mice, 20.558 ± 0.359 g. All other available studies with weight data examined gender-mixed groups in a different number of organs.
It can be assumed that, especially in the lung and liver tissue of Npc1 −/− mice, the sometimes massive intracellular deposits of myelin-like inclusions seen in electron micrographs ( Figure 17A,C) were responsible for the relatively higher organ weights of the mutants. was 26.601 ± 0.535 g and in female Npc1 +/+ mice, 20.558 ± 0.359 g. All other available studies with weight data examined gender-mixed groups in a different number of organs.
It can be assumed that, especially in the lung and liver tissue of Npc1 −/− mice, the sometimes massive intracellular deposits of myelin-like inclusions seen in electron micrographs ( Figure 17A,C) were responsible for the relatively higher organ weights of the mutants.  A conspicuously gender-different result was found for the weights of the adrenal gland. In males, the None group Npc1 −/− had weights in the range of the respective wild-types (Table 1, Figures 7A and 18A). One reason could be the clear occurrence of intracellular and extracellular myelin-like deposits in Npc1 −/− mice ( Figure 19A,E), not found in the respective wild-types ( Figure 19B,D). However, in female Npc1 −/− mice of the None group, the organs were significantly lighter than in the respective female Npc1 +/+ mice ( Figures 7B and 18A).
A conspicuously gender-different result was found for the weights of the adrenal gland. In males, the None group Npc1 −/− had weights in the range of the respective wildtypes (Table 1, Figures 7A and 18A). One reason could be the clear occurrence of intracellular and extracellular myelin-like deposits in Npc1 −/− mice ( Figure 19A,E), not found in the respective wild-types ( Figure 19B,D). However, in female Npc1 −/− mice of the None group, the organs were significantly lighter than in the respective female Npc1 +/+ mice (Figures 7B and 18A).   Concerning the femur lengths, it can be seen in Figure 18B that mice with smaller body weights generally had shorter femurs, irrespective of gender and genotype. Femur lengths of the four None groups were found in the identical descending order as the respective body weights: male Npc1 +/+ mice, female Npc1 +/+ mice, male Npc1 −/− mice, and female Npc1 −/− mice. The femur, as a representative of a long limb bone, appears to be primarily dependent on body weight for its length and not further dependent on cholesterol metabolism.

Relative Organ Weights of Npc1 +/+ and Npc1 −/− Mice of the None Groups Partly Differed in Both Genders
The relative organ weights of the None groups in the Npc1 −/− mice, calculated as percent of the respective body weights, differed considerably between organ and gender from the wild-types. In males, the relative weights of the heart, kidney, and bladder did not differ between Npc1 +/+ and Npc1 −/− mice; in females, besides the heart, kidney, and bladder, it was between the spleen and adrenal glands also.Other organs of Npc1 −/− mice had a higher or lower relative weight compared with Npc1 +/+ mice: in male Npc1 −/− mice, relatively higher weights were found in the lung, liver, spleen, adrenal gland, and testis, and only in the lung and liver in females. Relatively lower weights were found in Npc1 −/− males in vesicular and scent glands and in females in the ovary and uterus. It can be speculated that the organs which have relatively higher organ weights in Npc1 −/− mice contain lipid deposits that were not found in wild-types. With the exception of the testes, all other genital organs of both genders studied (vesicular and scent glands, ovary, uterus) had disproportionately small weights. One reason could be the massive disturbances of the sexual hormone system, depending on the starting product cholesterol [59,[123][124][125][126][127][128][129][130].

Absolute Organ Weights of Npc1 +/+ Mice Were Mostly Left Unchanged in Both Genders by the Applied Drugs
Administration of the various drugs to Npc1 +/+ mice resulted in small but significant changes in organ weight compared to the None group. In male mice, MIGLU induced an increased bladder weight (+13.8%), whereas COMBI (−4.7%) and HPßCD (−5.4%) reduced femur length. Even fewer organs were affected by the drugs in Npc1 +/+ females: MIGLU induced a decreased adrenal gland weight (−20.8%), and following HPßCD1x the kidney (−18.0%) and the uterus were lighter (−35.7%). It can be inferred that the drug-induced changes in cholesterol metabolism had only limited effects on normal mouse development.

Absolute Organ Weights of Npc1 −/− Mice Were Partly and Differently Increased in Both Genders by the Applied Drugs
Regardless of which drugs were used, the body weights of male and female Npc1 −/− mice increased significantly ( Figure 1) compared with the None groups. In Npc1 −/− mice of both genders, the absolute organ weights were never decreased by the applied drugs ( Figure 20). All drugs generally improved the health of these mice by at least in part disrupting the pathological cholesterol metabolism. The effects of the various drugs on the weights of the investigated organs, however, were quite different.   [40]. Tanaka et al. [66] described that chronic treatment with 1000, 2000, or 4000 mg/kg HPßCD1 (subcutaneously, once a week) significantly improved the survival of Npc1 −/− mice. Ramirez et al. [68] demonstrated that only weekly treatment with 4000 mg/kg) s. c. HPßCD prevents hepatosplenomegaly in Npc1 −/− mice. Moreover, Lopez et al. [103] showed that systemic administration of HPβCD, starting in early neonatal life, diminishes unesterified cholesterol accumulation in most organs, slows disease progression, and extends lifespan. Except for a slightly increased body weight, in no absolute organ-specific measurement of male and female Npc1 −/− mice, did HPßCD1x treatment have a significant effect ( Figure 20). It can be speculated that a single dose of HPßCD at P7 is too little to influence these parameters. This partly contradicts the results of Liu et al. [4,64] found in gendermixed mice groups, who state that administration of a single dose of HPßCD1x at P7 increased the lifespan of Npc1 −/− mice, and at P49 still reduced cholesterol in the liver, kidney, and spleen.
Chronic treatment with HPßCD, however, had a massive effect (Figures 20 and 21) started at P7 (Figure 22). Our data agree well with the results of Davidson et al. (2009), describing a significantly improved health status in the NPC1 disease mouse model. HPßCD significantly limits cholesterol levels and ganglioside storage in neurons of young Npc1 −/− mice [40]. Tanaka et al. [66] described that chronic treatment with 1000, 2000, or 4000 mg/kg HPßCD1 (subcutaneously, once a week) significantly improved the survival of Npc1 −/− mice. Ramirez et al. [68] demonstrated that only weekly treatment with 4000 mg/kg) s. c. HPßCD prevents hepatosplenomegaly in Npc1 −/− mice. Moreover, Lopez et al. [103] showed that systemic administration of HPβCD, starting in early neonatal life, diminishes unesterified cholesterol accumulation in most organs, slows disease progression, and extends lifespan.   With the exception of absolute stomach volume and femoral length, which showed no significant drug-related changes, all other organs increased their weight in at least one sex as part of one therapeutic scheme ( Figure 22). COMBI application increased weights in six male and six female organs, MIGLU in four males and two females, and HPßCD in four male and three female ones ( Figure 20). More than half of the male and female organs in which COMBI had a beneficial effect (n = 12) were also benefited by HPßCD treatment (n = 7). Thus, it can be assumed that the greater part of the COMBI effect is due to HPßCD.

Relative Organ Weights of Npc1 −/− Mice Were Differently Influenced in Both Genders
Studying the weights of organs in relation to body weight is often used in the Npc1 literature. It gives a reliable measurement to indicate whether an organ is smaller, equal, or larger in relation to body weight [54,92,94,97,98,110,114] (Supplementary Materials  Table S1). Organs that lost or gained relative weight appeared to be affected by treatment differently than the whole animal.
Taking male and female Npc1 −/− mice together, in the 92 organ measurements, the relative weights in 60 cases were not significantly different from the None groups ( Figure 21). For 22 measurements, the relative organ weights decreased, and for 10, they increased compared with the None groups ( Figure 21). With the exception of absolute stomach volume and femoral length, which showed no significant drug-related changes, all other organs increased their weight in at least one sex as part of one therapeutic scheme ( Figure 22). COMBI application increased weights in six male and six female organs, MIGLU in four males and two females, and HPßCD in four male and three female ones ( Figure 20). More than half of the male and female organs in which COMBI had a beneficial effect (n = 12) were also benefited by HPßCD treatment (n = 7). Thus, it can be assumed that the greater part of the COMBI effect is due to HPßCD.

Relative Organ Weights of Npc1 −/− Mice Were Differently Influenced in Both Genders
Studying the weights of organs in relation to body weight is often used in the Npc1 literature. It gives a reliable measurement to indicate whether an organ is smaller, equal, or larger in relation to body weight [54,92,94,97,98,110,114] (Supplementary Materials Table S1). Organs that lost or gained relative weight appeared to be affected by treatment differently than the whole animal.
Taking male and female Npc1 −/− mice together, in the 92 organ measurements, the relative weights in 60 cases were not significantly different from the None groups ( Figure  21). For 22 measurements, the relative organ weights decreased, and for 10, they increased compared with the None groups ( Figure 21). Because we are not aware of any study that has published the effects of the drugs used in Npc1 −/− mice on organ-specific weights in a gender-specific manner, the publications cited in this paragraph refer to gender-mixed mice. Comparable to Figure 21, Ebner et al. [54] showed that both COMBI and HPßCD monotherapy (injected weekly with HPβCD (4000 mg/kg starting at P7) significantly reduced the liver-to-body-weight (LW/BW) ratio in Npc1 −/− mice and reached the values found in sham-treated Npc1 +/+ mice.
In the study of Lopez et al. [103], Npc1 −/− mice and their Npc1 +/+ controls were given four weekly subcutaneous injections of either saline or HPβCD (4000mg/kg bw) from 49 days of age and studied at P77. When compared with their Npc1 +/+ controls at P49, Npc1 mutant mice exhibited the prototypical relative organomegaly of the liver, spleen, and lung. The enlargement of the liver seen in the 49-day-old Npc1 −/− mice persisted in the P77 mutants given saline but was substantially diminished in their counterparts receiving HPβCD. The changes in relative spleen weight paralleled those of the liver, whereas relative kidney weights did not change with HPβCD treatment. In the case of the lungs, relative weights were consistently greater in the Npc1 −/− mice but otherwise did not change as a function of age or treatment. Although Lopez et al. [103] used a late-onset therapy, their results tend to agree with ours.
Neßlauer et al. [116] analyzed the organ-to-body weight ratio in the spleen (SW/BW) after COMBI treatment. Consistent with our results, their mutant Npc1 −/− mice showed increased spleen weight and increased lipid accumulation that could have been avoided by COMBI treatment.
Ramirez et al. [68] injected Npc1 +/+ and Npc1 −/− mice with saline or a dose of HPβCD (4000 mg/kg) subcutaneously at P7 and every week thereafter until mice were studied as young adults at P49. The Npc1 −/− mice that received saline alone exhibited significant enlargement of the liver, spleen, and lung but not of the other organs so far analyzed. Weekly treatment with HPβCD prevented this hepatosplenomegaly. We found comparable results in these three organs but very different results in many other organs after using different treatment strategies (Figures 20 and 21). The majority of organs did not show significant drug-induced changes in their relative weights. This means that these organs changed their weight proportionally to the respective body weight, independent of the treatment used. Drug effects were comparable in these organs and throughout the body. Correspondingly, larger organs were found, for example, in the hearts and lungs of heavier Npc1 −/− mice. Concerning the lungs, Lopez et al. [103] showed in the NPC mouse model that, even after systemic administration of HPβCD, the relative lung weights were consistently greater in the Npc1 −/− mice. They did not change as a function of treatment. According to our results, the same held true for the heart in all treatment groups.

Relative Organ Weights of Npc1 −/− Mice Were Significantly Decreased in 22 Cases
Differentiated results were found in the soft tissue organs liver, spleen, kidney, and adrenal gland, which had relatively reduced organ weights in some drug-treated groups either in both sexes or in only one sex. Drug treatment in these organs resulted in decreased pathological lipid deposits, so the organs were lighter than expected, considering a proportionate organ and body growth.
H&E staining of Sham-treated, COMBI-treated, or mono-treated with HPβCD (4000 mg/kg starting at P7) Npc1 +/+ mice showed normal liver morphology and normal microvascular integration. In contrast, liver tissue from sham-treated Npc1 −/− mice showed necrosis. Moreover, the liver architecture was characterized by lipid accumulation in hepatocytes-analog to Figure 17C-and frequent invasion of histiocytic foam cells into sinusoids. Following COMBI therapy and monotherapy with HPβCD (4000 mg/kg starting at P7), Npc1 −/− mice showed an improvement in liver morphology and less necrosis but still some fat deposits [54].
Lopez et al. [103] also found large numbers of foamy, lipid-laden macrophages in the livers of Npc1 −/− mice who were given saline compared with their Npc1 +/+ littermates. In contrast, in the 91-day-old mice administered with HPβCD, there was a significant reduction in the presence of these macrophages [103].
H&E staining of Sham-treated or COMBI-treated Npc1 +/+ spleen showed normal morphology and a regular lymphoid follicular architecture. In contrast, spleen tissue from sham-treated Npc1 −/− mice showed significant morphological differences due to the infiltration of foam cells, which strikingly alter the splenic architecture by displacing the lymphoid follicles. In addition, this phenomenon was remarkably reduced in COMBI-treated Npc1 −/− mice showing fewer foam cells, generally resembling the Npc1 +/+ phenotype [116].
Ramirez et al. [68] showed, in a multi-organ study, that in untreated P49 Npc1 −/− mice, numerous lipid-laden macrophages were scattered throughout the liver. At the same time, this infiltrate is almost completely absent in HPβCD-treated animals. Even after 160 days of treatment, the architecture of the liver in Npc1 −/− mice was essentially normal, apart from occasional accumulations of macrophages in a pericentral distribution. Histologically, the liver was completely normal in Npc1 +/+ animals treated with weekly HPβCD. However, the lungs behaved differently. In the P49, untreated Npc1 −/− mice displayed small clusters of macrophages scattered throughout the alveoli of the lungs-as seen in the electron micrograph Figure 17A-and similar clusters were still found after treatment with HPβCD. This progressive infiltration continued even with weekly HPβCD treatment until, at P160, accumulations of lipid-laden macrophages filled many of the alveolar spaces. Notably, the lungs appeared completely normal in the Npc1 +/+ mice treated weekly with HPβCD. Finally, renal architecture was essentially normal in the untreated Npc1 −/− mice, but vacuolation was noted in some tubular epithelia after administration of HPβCD, as previously reported [68].
In line with histological results showing lower drug-induced lipid storage in various organs, weight analysis revealed lower relative organ weights for the liver, spleen, and adrenal gland after MIGLU, HPβCD, and COMBI.
The significant decrease in relative femoral lengths in all four drug-treated groups of both sexes compared with the None groups is likely caused by constant femur measurements in these mice in combination with significantly increased drug-induced body weights. The organs belonging to the group of secondary sex organs, with the exception of the bladder in the Npc1 −/− COMBI-treated males, increased their relative weight significantly. It can be speculated that a partly drug-related normalization of steroid hormone levels increased the growth and development of these hormone-dependent organs in particular. It is well known that the steroid hormone levels of the Npc1 −/− None groups significantly decrease compared with the wild-types [59,[123][124][125][126][127][128][129][130]. However, it must be noted that the verification of a hypothetical drug-induced increase in steroid hormone concentrations in Npc1 −/− mice is still pending.

Drug-Specific Effects on Absolute and Relative Organ Weights of Male and Female Npc1 −/− Mice
Comparing the significant effects of the various treatments on organ weights of Npc1 −/− mice, it can be summarized that, concerning absolute weights, the COMBI effects were seen in 12 (6 in males, 6 in females), the MIGLU effects in 6 (4 in males, 2 in females), and the HPβCD effects in 7 (4 in males, 3 in females) measurements ( Figure 20). Concerning relative organ weights, significant changes were found in 11 measures (8 in males, 3 in females) after COMBI, 9 (6 in males, 3 in females) following MIGLU, and 8 (5 in males, 3 in females) following HPβCD treatment ( Figure 21).

Organ Weights of Npc1 −/− Mice Depend on Various Parameters and Are Partly Influenced by Pharmacological Treatment Approaches
Specific organ weights are the result of a complex interplay of various parameters. Primarily, organ weight is dependent on body weight: heavier mice have heavier organs. Weight is also influenced by the sex of the mouse: females have smaller organs than males, possibly due to their smaller body weight and/or hormonal status. The mutant Npc1 gene affects weight by intracellular accumulation of unesterified cholesterol, sphingomyelin, phospholipids, glycosphingolipids, and GM2 and GM3 gangliosides in late endosomes/lysosomes, endoplasmic reticulum and the Golgi apparatus. The extent of the deposits varies from organ to organ, and so do the specific organ weights. Because the metabolism of cholesterol as the precursor for the synthesis of sex hormones is massively disturbed, steroid hormone levels of Npc1 −/− mice are significantly decreased compared with wild-type, resulting in hampered organ development and weight. Interventions in cholesterol metabolism by MIGLU, HPβCD, and COMBI treatments have a positive effect on clinical symptoms in Npc1 −/− mice, leading to reduced lipid accumulations in organs and to a still hypothetical drug-induced increase in steroid hormone concentrations in Npc1 −/− mice. It is hypothesized that the decreased relative weights of thoracic and abdominal organs are due to drug-induced decreased lipid accumulation. The increased relative weights of sex organs are due to the drug-induced normalization of steroid hormone concentrations.

Animals
All animal procedures were approved by the local authorities (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei des Landes Mecklenburg-Vorpommern; approval ID: 7221.3-1.1-030/12, 14 June 2012). All institutional guidelines for animal welfare and experimental conduct were followed, and all efforts were made to minimize suffering.
Heterozygous Npc1 +/− mice breeding pairs of Npc1 mice (BALB/cNctr-Npc1 m1N /-J) were obtained from Jackson Laboratories (Bar Harbor, ME, USA) for generating homozygous Npc1 −/− mutants and Npc1 +/+ control wild-type mice. Experimental animals were maintained under standard conditions with free access to food and water with a 12 h day/night cycle, a temperature of 22 • C, and a relative humidity of about 60%. Genotypes were determined by postnatal day P7 by PCR analysis of tail DNA as previously described [135,136]. Npc1 −/− mutants and Npc1 +/+ wild-type controls of both sexes were used for different therapeutic treatment schedules. Fixed cadavers, still containing most organs, from different studies were collected from 2012 to 2019. Altogether, 176 wild-type mice (103 females, 73 males) and 175 mutant mice (92 females, 83 males) were involved in this study. The exact numbers of animals investigated in the various groups are listed in Table 2. None (None group): These mice were left uninjected. All mice were sacrificed at P65.

Body Weight and Anesthesia
Before sacrification, body weights were measured, and animals were deeply anesthetized with an in-house drug mixture, a diluted solution of 0.75 g ketamine hydrochloride (contained in 7.5 mL of a 10% ketamine hydrochloride ready-to-use preparation; Ketamin ® 10%, Bela-Pharm, Vechta, Germany) + 0.05 g xylazine (contained in 2.5 mL of a 2% xylazine ready to use preparation; Rompun ® , Bayer, Leverkusen, Germany), and 90 mL saline.

Dissection of the Animals
First, the heart was separated from the lungs and removed with forceps. Thereafter, the lungs were separated from the thorax with a thin spatula. After the dissection of the stomach and spleen, the liver was removed. The kidneys and adrenal glands were then dissected. In female mice, first, the ovaries and then the uterus were separated. In male mice, first, the scent glands were exposed, followed by the removal of the vesicular glands and the testes/epididymites. Thereafter, the bladder was dissected.
In the last step, the femora were exposed and disarticulated in the hip and knee joints.

Determination of the Organ Weight, Size, and Volume
The organ weights were determined using a fine scale (device GR202, accuracy 0.000(0) g A&D Europe GmbH, Darmstadt, Germany). For this purpose, the organs were removed from the PFA solution and dried briefly on filter paper. In addition, the length of the femora was measured with a caliper, and the volume of the stomach was determined from its length, width, and height.

Electron Microscopy
After perfusion with 0.1 M phosphate buffer containing 2.5% paraformaldehyde, six Npc1 −/− and Npc1 +/+ mice were postfixed in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde for at least 24 h at 4 • C. Subsequently, the adrenal glands, lung, and liver were excised and kept in the same fixative. Thereafter, the specimens were osmicated, washed, dehydrated through a graded series of ethanol, and embedded in Epon 812 (Plano GmbH, Marburg, Germany). Ultrathin sections (about 70 nm) were mounted on pioloformcoated slot copper grids and contrasted with uranyl acetate (4 min), followed by lead citrate (2 min). The specimens were examined with a Zeiss EM 902 transmission electron microscope (Zeiss, Oberkochen, Germany) at 80 kV. Photographs were taken using a CCD camera (Proscan, Lagerlechfeld, Germany) and adjusted using Photoshop CS2 software (Adobe Systems).

Data Analysis
The results are presented as means ± SEM. In general, an overall significance level of p = 0.05 was used. All data were subjected to three-or two-way ANOVA. In the case of statistically significant different mean values, the Holm-Sidak approach was used for post hoc comparisons. All statistical analyses were conducted using SigmaPlot 14 Software (Systat Software, Inc., San Jose, CA 95110, USA).

Conclusions
The combination therapy of miglustat as a substrate reduction agent (inhibitor of the glucosylceramide synthase) in combination with the sterol chelator 2-hydroxypropyl-ßcyclodextrin (reverser of the cholesterol transport defect) seemingly had an additive effect on the normalization of the cholesterol metabolism and, subsequently, on the organ weight outcomes in Npc1 −/− mice, especially in the liver, spleen, adrenal gland and genital organs. The comparison of both genders reveals that for all drug effects of 92 measures taken together, absolute weights were changed in 14 cases in males and 11 in females. Respective relative weights significantly changed in 19 of the measurements taken in males and 9 in females. Remarkably, male Npc1 −/− mice were more sensitive to drug treatment.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.